JP6701338B2 - Biochemical/chemical assay device and method with simplified steps, fewer samples, faster speed and easier to use - Google Patents

Description

CROSS REFERENCE This application is related to US Provisional Application No. 62/202,989 filed on Aug. 10, 2015, US Provisional Application No. 62/218,455 filed on Sep. 14, 2015, Feb. 9, 2016. Provisional Application No. 62/293,188 filed on March 8, 2016, Provisional Application No. 62/305,123 filed March 8, 2016 and Provisional Application No. 62/ on July 31, 2016. No. 369,181, all of which are hereby incorporated by reference in their entireties for all purposes.

FIELD The present invention relates to the field of biochemical/chemical sampling, sensing, assays, and applications.

Background In many biochemical/chemical sensing and testing (eg, immunological assays, nucleotide assays, hemocytometry, etc.), chemical reactions, and other processes, processes (eg, reagent binding, mixing, etc.) It can speed up and quantify parameters (eg, analyte concentration, sample volume, etc.), simplify the sampling and measurement process, can handle small volume samples, and can assay the entire assay. The process can be performed in less than 1 minute, the assay can be performed by a smartphone (eg mobile phone), the non-specialist can perform the assay himself, and the test results can be related locally, remotely or wirelessly. What is needed is a method and apparatus that enables a person to communicate. The present invention relates to methods, apparatus and systems that address these needs.

  The following summary is not intended to include all features and aspects of the present invention. The present invention provides faster and more biochemical/chemical sensing (including but not limited to immunological assays, nucleic acid assays, electrolyte analysis, etc.) over many conventional sensing methods and devices. Methods, devices that are more sensitive, have fewer steps, are easier to perform, require less sample, have less or less need (or no) for professional assistance, and/or have lower costs , And the system.

  The goal of many current laboratory tests is to accurately determine the absolute concentration of an analyte in a sample. For example, the RBC test involves counting the number of red blood cells in a defined volume of whole blood and then calculating the number of red blood cells per microliter of whole blood. However, such measurements typically require special instruments and/or accurate measuring devices (such as accurate pipettes) that can accurately measure relatively small volumes of biological fluids, and thus require the use of specialized testing centers. If not (ie, "at home", "at the pharmacy", or in a "clinical setting" environment), it may be difficult to perform.

Volume Measurement of Relevance Many assays provide the absolute concentration of an analyte in a sample. However, if only a small volume (eg 100 nL-10 μl) is analyzed, the results of such an assay will be very inaccurate. This is because it is difficult to dispense and/or measure accurately in a small volume.

In some assays, liquid samples can be placed between two plates separated by a spacer and analyzed. Theoretically, the volume of the analyzed sample can be calculated by multiplying the area of the analyzed sample by the thickness of the analyzed sample. However, in practice, such an estimation is not easily done and is very inaccurate due to various causes. By way of example, some devices use beads to space the plates, and the beads or one of these plates is deformable. Such devices are prone to inaccuracy for the following reasons.
• The spherical spacers have a much smaller contact area with these plates (almost one point). In such a device, due to the much smaller contact area, the pressure exerted on the contact area between the plate and the sphere is much greater for the pressing force applied per unit. The large pressure causes the spheres and/or plates (if they are flexible) to deform, distorting any measurements.
The spherical spacers are usually randomly distributed between the two plates. Since the spherical spacers are randomly distributed, the distance between the spacers fluctuates greatly and some distances are quite large. This causes the spacers and/or plates (if they are flexible) to deform much more in some areas compared to other areas and distort the results as well.
• Randomly placed spacers in close proximity to each other can interfere with the movement of the analyte (eg, cells) and thus cause more difficulties for the analyte or cells. May create "lumps".
-A large deformation of one of these plates can lyse the cells and can cause errors in the cell counting task.
The volume calculation is inaccurate, as the number of spherical spacers in the area to be analyzed and the extent to which the spacers and/or one of these plates are deformed vary from sample to sample.
-The deformation causes a variation in the time required for the molecules to diffuse to the surface of one of these plates.

  In a device with a spherical spacer, the volume of a portion of the analyzed sample is a) counting the spheres in the volume of the analyzed sample, and b) experimentally estimating the thickness of one layer of the sample ( This can potentially be estimated by, for example, adding an internal standard such as an immiscible liquid containing a known concentration of calibrator that can be used to calculate the distance between plates). However, these extra steps are inconvenient to perform and the measurements obtained from such devices are still not very accurate, as the top plate and/or the spacer will be significantly deformed during use.

  In contrast, embodiments of the methods and apparatus of the present invention have substantially uniform height, substantially uniform cross-section (eg, pillars with straight sidewalls) and planar (ie, “flat”). It depends on spacers fixed to one or more of these plates in a regular pattern with tops spaced apart from each other by a constant and defined distance (i.e. not random positions according to Poisson statistics). During use of some embodiments of the method and apparatus, the spacers and plates do not significantly compress or deform in any dimension, and at least while the plates are in the closed position and pulled by capillary forces. Or does not deform. The device makes it very easy to use in the use of this device the volume of the sample for which data is acquired (ie the “relevant volume”, or the volume of the sample in the area to be analyzed) It can be calculated accurately, and in some cases even unknown amounts of sample can be calculated before starting the assay, even if deposited on the device. In the closed position, the plate is substantially flat (meaning that the sample thickness is uniform) and the number and dimensions of the spacers in the analysis area are known, so that the volume of the sample in that area is After performing an assay that can be easily calculated with high accuracy, the relevant volume sample can be determined without the need to count spacers in the region or to estimate further sample thickness. It is also necessary to deposit a certain amount of sample in the device. Moreover, at the beginning of the incubation, the analyte molecules should be evenly distributed (to the extent that Poisson statistics allow) over the relevant volume rather than being concentrated in one region compared to another.

Short Reaction Times Known assays, but due to the very low diffusion constants of many analytes in an aqueous environment, many assays require long incubation times (always hours, sometimes days), agitation, and mixing. It requires the use of promoting agents or forces. Such assays are designed to allow the analytes to diffuse laterally from an initial position on one of these plates to a remote location (eg, Wei et al., Nucl. Acids Res. 33: e78 and Toegl et al. J. Biomol. Tech. 2003 14:197-204). Such systems are limited because it can take several hours to get the results. Furthermore, when the results are obtained, it is usually hard to say for sure that the reaction has reached equilibrium at the end of the reaction. In particular, this uncertainty makes it impossible to estimate the absolute concentration of the analyte in the sample.

  As described in detail below, in some embodiments of the present methods and devices, the spacer height and assay endpoint are selected to limit the lateral diffusion of the analyte during the assay. You can In these cases, such an assay (typically a binding assay) can be performed in a very short time. Also, the concentration of the analyte in the sample can be very accurately estimated, whether the entire sample is unanalyzed or of unknown quantity.

  In these embodiments, i. The time required for the target entity in the closed configuration to diffuse across the thickness of a layer of uniform thickness (i.e., more than the time it takes for the analyte to diffuse vertically from one plate to another). Short time), and ii. Time shorter than the time it takes for the target entity to diffuse laterally over the linear dimension of a given area of the binding site (ie, the time it takes for the analyte to diffuse laterally from one side of the binding site to the other). In less time, the assay can be stopped and/or the assay results read. In such a “locally coupled” configuration, the volume of the part of the sample for which data is acquired (“relevant volume”) is the volume of the sample that is directly above the analysis area, which is rational and accurate. Can be estimated. In fact, the volume of the part of the sample from which the data was taken can be known before the start of the assay. Such a "local binding" embodiment is one in which the binding between any analyte and/or detection reagent causes the sample and any detection reagent to be pressed as a thin layer overlying the binding site. It has the additional advantage of the embodiment not pressed as a thin layer that equilibrium is reached more quickly, for example when a drop of sample is simply placed on top of the plate with the binding sites. As such, binding equilibrium is often reached in seconds rather than minutes, and thus many assays, especially binding assays, can be completed very quickly, eg, in less than 1 minute.

Multiplexing The "local binding" configuration also allows multiple assays to be performed without fluidly isolating different reactions from each other. In other words, multiple assays can be run in an open environment where the assays are not isolated from each other (ie, no fluidic isolation). For example, in a local binding embodiment, two different analytes in the same sample can be assayed in parallel, stopping the assay before the analytes diffuse from one assay area to another. And/or by reading the assay results, the absolute concentration of each of these analytes in the sample can be determined even though the analytes in the sample are not in fluid isolation from each other.

  Multiple assays can be performed on one sample without fluidic isolation by simply sandwiching the sample between two plates and performing the assay in a diffusion limited manner, with some advantages. There is. For example, simply drop a drop of a sample of unknown volume (eg, blood), spread the sample onto the plate by pressing the sample and plate together, and incubate the sample for a period of time to allow for multiple sites within the device. The assay can be completed by reading from. When performing such a method, it is not necessary to transfer a defined amount of sample to some chambers, which is difficult to perform without accurate fluid transfer and/or metrology equipment. Note that the assay is very rapid for the reasons mentioned above. Furthermore, the device is easy to manufacture, since it is not necessary to manufacture the plate with "walls". Finally, neither of the plates requires a port, i.e. a port potentially used for addition or removal of sample or reagents while the device is in the closed position.

Amplification Surface Also, in some embodiments of the present devices and methods, the device can include an “amplification surface”, eg, referencing a surface that enhances a signal such as fluorescence or luminescence generated by a detection reagent. .. In some cases, the signal can be enhanced by nanoplasmonic effects (eg, surface-enhanced Raman scattering). Li et al., Optics Express 2011 19: 3925-3936 and WO 2012/024006 describe examples of signal enhancement by an amplification surface and are incorporated herein by reference. In some cases, the amplification surface may be a disk-coupled dot-on-pillar antenna array (D2PA), which is described in US Pat. No. 9,013,690. In use, a device containing an amplification surface can amplify the signal by a factor of 10 ³ or more, compared to a detector that is not configured to enhance the signal, thus enabling analyte detection with very high sensitivity. To In some embodiments, digitally counting the amount of an analyte in a sample of relevant volume, particularly a non-cellular analyte detected using a sandwich assay, using, for example, the method described in WO2014144133. You can The use of an amplification surface optionally allows the assay to be read using a smartphone or the like.

Other Features In an embodiment of the device, when the plates are submerged in an aqueous environment, the position of spacers secured to one or more plates is not changed or the spacers are not washed away. The spacer is not spherical and is not fixed to the surface of the plate by a weak force such as electrostatic force or gravity. In some embodiments, the plate with spacers may be monolithic. In many embodiments, the spacer is not prefabricated and then affixed (eg, affixed, etc.) to the plate. Rather, spacers may be grown and/or etched on the plate using embossing and/or microfabrication (eg, photolithography) processes.

  In the closed position, the parameters of the spacer (eg, such that the top plate, which may be flexible) is not significantly deformed over the part of the sample being analyzed (the “relevant volume” of the sample). Their cross-section, spacing, density, etc.) can be optimized. In some cases, the spacer parameters can be adjusted by the flexibility of the top plate. For example, if the top plate is more flexible, the spacers can be closer together. Similarly, if the top plate is less flexible, the spacers may be farther apart.

  Further, in use of many embodiments of the device, analytes do not move directionally through the device after the device is closed. Thus, in the closed configuration, analytes are not sorted or fractionated, as described in Austin (US Pat. No. 6,632,652), and the analytes (eg, by gravity or electrophoresis). It may not flow forcedly and directionally through the device. In many cases, it is not necessary to couple the device to a power source to generate electromotive force. In many embodiments, the analyte is concentrated in one area relative to another, as there are no "obstacles" that interfere with the passage of the analyte (cell) while the sample is being spread. Instead, it is evenly distributed (to the extent that Poisson statistics allow) over the relevant volume. Furthermore, in other devices, the function of the cover plate is to seal the device to prevent leakage of liquid, so that if neither plate has sample, the cover plate should be placed on top of the substrate. be able to. Such devices do not push liquid onto open plate surfaces to produce a thin layer of sample that can be analyzed. Also, in other devices, an important function of the pillars is to “filter” or sort nanoparticles (eg, cells, etc.). Therefore, the distance between the pillars is determined by the sorted nanoparticles, not for the purpose of uniform spacing between the cover plate and the substrate. Finally, in many devices, such as the Austin device, the accuracy of the sorting is controlled primarily by the distance between the columns, not by the distance between the plates, and control of the distance between the plates is not important. I think that the. Accordingly, such disclosure does not alter the plate spacing uniformity by changing the pillar size, shape, spacing between the pillars, and the like.

  In view of the above, the present apparatus and methods are easy to use, inexpensive, easy to manufacture, and very fast for multiple analytes in a liquid sample (or multiple, if the apparatus and methods are implemented in multiplex fashion). Analyte).

  One aspect of the invention is the use of a pair of mutually movable special plates for a smaller volume of sample or one or more reagents, or both, for a simpler, faster and/or better assay. Is a means for operating. The operations include, but are not limited to, sample reformation, forced sample flow, sample-reagent contact, sample volume measurement, shortened diffusion distance, increased collision frequency, and the like. Has a beneficial effect on a particular assay. In the present invention, the special features and characteristics on the plates, the special methods for handling the plates, and the special methods for handling the reagents and samples provide advantages in the assay.

  One aspect of the invention is a means for forming at least a portion of a small droplet of a liquid sample attached to a plate into a thin film having a controlled, predetermined, and uniform thickness over a wide area. The uniform thickness may be as thin as less than 1 μm. Moreover, the present invention allows the same uniform thickness to be maintained for a long time without evaporation to the environment.

  Another aspect of the present invention is a means for determining a partial or total volume of a sample utilizing the predetermined uniformly thin sample thickness formed by the present invention without the use of a pipette or the like.

  Another aspect of the invention is one embodiment of a spacer (for controlling the spacing between two plates) having a pillar shape with a flat top and a substantially uniform cross section. Such spacers offer many advantages in controlling sample thickness over ball (bead) spacers.

  Another aspect of the invention is multiple embodiments of spacers (for controlling the spacing between two plates) having a pillar shape with a flat top and a substantially uniform cross section. Such spacers offer many advantages in controlling sample thickness over ball (bead) spacers.

  Another aspect of the invention is the use of a particular chemical reaction (or mixing) primarily in only a small portion of the sample, but not in the other parts of the sample, without the use of fluidic separation between the two parts of the sample. ) Is a means to generate.

  Another aspect of the invention is the use of multiple chemical reactions (or mixing), predominantly only in a small portion of the sample, but not in other parts of the sample, without the use of fluidic separation between different parts of the sample. Is a means to generate. Thus, the present invention allows multiple assays to be run in parallel with small drops of sample without fluidic separation between different reaction sites.

  Another aspect of the invention is a means of making assays (eg, immunological assays, nucleic acid assays, etc.) more rapid. For example, the saturation incubation time (the time for intermolecular binding to reach equilibrium) is reduced from a few hours to less than 60 seconds.

  Another aspect of the invention is a means of significantly increasing detection sensitivity by one method or a combination of methods, including an amplification surface, large or bright labels, and the like.

  Another aspect of the invention is a means to perform the assay using a very small sample volume, eg 0.5 μL (microliter) or less.

  Another aspect of the invention is a means of simplifying the assay by allowing minute body fluids to be deposited directly from the subject onto the test or sample area.

  Another aspect of the invention is a means to simplify and speed up the assay by pre-coating the plate with reagents. For example, the plate is pre-coated with the capture agent and the detection agent and dried. Another example performs detection by pre-coating the plate with all the necessary detection reagents and attaching the sample to the pre-coated plate without the need to deposit other reagents.

  Another aspect of the invention is a means for performing an assay reading by cell phone.

  Another aspect of the invention is a means that allows direct instillation of one's body fluid (eg saliva) between a pair of plastics to allow one's own biomarker test in less than 60 seconds.
[Invention 1001]
(A) obtaining a sample containing the analyte,
(B) obtaining a first plate and a second plate that are moveable relative to each other in different configurations, each plate having a substantially flat sample contact surface; One or two plates are flexible, one or two of said plates comprising spacers fixed to their respective sample contact surfaces, said spacers having a predetermined substantially uniform height, and A step having a predetermined constant distance between spacers that is at least about twice as large as the analyte size and up to 200 μm (micrometers);
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration comprising the two plates being partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) after (c), compressing at least a portion of the sample with the two plates into a layer of substantially uniform thickness, the uniform thickness of the plates Limited by the sample contact surface of the, the uniform thickness of the layer is adjusted by the spacer and the plate, and the compression is
Uniting the two plates, and
In order to press the plates together into the closed configuration, in parallel or in sequence, one or more regions of at least one of the plates are suitably pressed, and the appropriate said press is applied to the at least part of the sample. A substantially uniform pressure on the plate is generated such that the pressing causes the at least a portion of the sample to spread laterally between the sample contacting surfaces of the plate and the closed configuration to provide a uniform thickness region. The space between the plates in the layer is adjusted by the spacer.
Including steps, and
(E) analyzing the analyte in the layer of uniform thickness while the plate is in the closed configuration.
A method for analyzing a liquid sample, comprising:
The suitable press is a method for making the pressure applied to a certain region substantially constant regardless of the shape change of the outer surface of the plate,
The method wherein the parallel press simultaneously applies pressure to a desired region and the sequential press applies pressure to a portion of the desired region and gradually transitions to another region.
[Invention 1002]
After step (d) and before step (e), the method further comprises removing a suitable pressing force after the plate is in the closed configuration, after removing the suitable pressing force. The thickness of the uniform thickness layer is (i) substantially the same as the thickness of the uniform thickness layer prior to removing the appropriate pressing force, and (ii) of the spacer The method of invention 1001, wherein the height deviates less than 10%.
[Invention 1003]
The method of any of the preceding inventions wherein the sample attachment in step (c) is direct attachment from the subject to the plate without the use of any transfer device.
[Invention 1004]
The method of any of the preceding claims, wherein the amount of the sample deposited on the plate is unknown during the deposition of step (c).
[Invention 1005]
The analyzing step (e) includes calculating a volume of the relevant sample volume by measuring a side area of the relevant sample volume, and calculating the volume from the side area and a predetermined spacer height. The method of any of the preceding inventions, comprising the step of:
[Invention 1006]
The analyzing step (e) includes
i. Luminescence selected from photoluminescence, electroluminescence, and electrochemiluminescence,
ii. Surface Raman scattering,
iii. Electrical impedance selected from resistance, capacitance, and inductance, or
iv. i. ~ Iii. Any combination of
A method according to any of the preceding claims, including the step of measuring
[Invention 1007]
The method of any of the preceding claims, wherein said analyzing step (e) comprises reading, image analysis, or counting said analytes, or a combination thereof.
[Invention 1008]
The sample comprises one or more analytes and the one or two plate sample contacting surfaces comprises one or more binding sites, each binding site binding to and immobilizing a respective analyte. , The method of any of the preceding inventions.
[Invention 1009]
One or two plate sample contact surfaces each include one or more storage sites for storing one or more reagents, said reagents being dissolved and diffused into said sample during or after step (c). , The method of any of the preceding inventions.
[Invention 1010]
One or two plate sample-contacting surfaces include one or more amplification sites, each of said one or more amplification sites provided that said analyte or said analyte label is within 500 nm of one amplification site. The method of any of the preceding claims, wherein the signal from the analyte or the label can be amplified.
[Invention 1011]
i. The one or two plate sample contacting surfaces include one or more binding sites, each said binding site binding to and immobilizing a respective analyte, or
ii. The one or two plate sample contacting surfaces each include one or more storage sites for storing one or more reagents, said reagents being dissolved and diffused into said sample during or after step (c). , The sample comprises one or more analytes, or
iii. Wherein the analyte or the analyte label is 500 nm from the amplification site, one or more amplification sites, respectively, can amplify the signal from the analyte or the label, or
iv. i. ~ Iii. Any combination,
Any of the methods of the invention above.
[Invention 1012]
The liquid sample is amniotic fluid, aqueous humor, vitreous humor, blood (eg, whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax, ear chyme, chyme, Endolymph, perilymph, feces, respiration, gastric acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, purulent fluid, mucous secretions, saliva, exhaled breath condensate, sebum, The method according to any one of the above-mentioned present inventions, which is a biological sample selected from semen, sputum, sweat, synovial fluid, tear fluid, vomitus, and urine fluid.
[Invention 1013]
The method of any preceding invention, wherein the layer of uniform thickness in the closed configuration is less than 150 μm.
[Invention 1014]
The method of any of the preceding claims, wherein the suitable pressing during step (d) is manual.
[Invention 1015]
The method of any of the preceding inventions, wherein the suitable press of step (d) is provided by a pressurized liquid, a pressurized gas, or a conformal material.
[Invention 1016]
The method of any of the preceding claims, wherein said analyzing step comprises counting cells within said layer of uniform thickness.
[Invention 1017]
The method of any of the preceding claims, wherein said analyzing step comprises conducting an assay in said layer of uniform thickness.
[Invention 1018]
The method of invention 1017, wherein said assay is a binding assay or a biochemical assay.
[Invention 1019]
The method of any of the preceding claims, wherein the total volume of the deposited sample is less than 0.5 μL.
[Invention 1020]
The method of any of the preceding claims, wherein multiple drops of sample are deposited on one or two of the plates.
[Invention 1021]
First plate and second plate
Including,
i. The plates are movable relative to each other in different configurations,
ii. One or two plates are flexible,
iii. Each said plate has a sample contact area on its respective surface for contacting a sample containing an analyte,
iv. One or two of the plates includes spacers affixed to respective sample contact areas, the spacers having a predetermined substantially uniform height and a maximum of at least about 2 times greater than the size of the analyte. Has a predetermined constant inter-spacer distance of 200 μm, and at least one said spacer is in said sample contact area,
One configuration is an open configuration in which the two plates are separated and the spacing between the plates is not adjusted by the spacer and the sample adheres to one or two of the plates, and
Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein at least a portion of the sample is compressed by the two plates to a very uniform thickness. An apparatus for analyzing a liquid sample, wherein the uniform thickness of the layer is limited by the sample contact surface of the plate and is regulated by the spacer and the plate.
[Invention 1022]
The device of invention 1021, wherein the distance between the spacers is in the range of 1 μm to 120 μm.
[Invention 1023]
The device of any of the preceding claims wherein the flexible plate has a thickness in the range of 20 m to 250 m and a Young's modulus in the range of 0.1 GPa to 5 GPa.
[Invention 1024]
An apparatus according to any one of the preceding inventions, wherein for the flexible plate, the product of the thickness of the flexible plate and the Young's modulus of the flexible plate is in the range of 60 GPa-μm to 750 GPa-μm.
[Invention 1025]
The layer of the sample of uniform thickness is at least 1 mm ² The device of any of the preceding inventions which is uniform over a side area that is
[Invention 1026]
The apparatus of any of the preceding claims wherein the layer of uniform thickness sample has a thickness uniformity of up to ±5%.
[Invention 1027]
The device of any of the preceding claims wherein the spacer is a column having a cross-sectional shape selected from a circle, polygon, ring, square, rectangle, oval, ellipse, or any combination thereof.
[Invention 1028]
The spacers have a pillar shape, have a substantially flat upper surface, and have a substantially uniform cross section, and for each spacer, the ratio of its lateral dimension to the height of the spacer is at least 1. The device of any of the preceding claims, wherein:
[Invention 1029]
The device of any of the preceding claims wherein the distance between the spacers is periodic.
[Invention 1030]
The apparatus of any of the preceding claims, wherein the spacer has a fill factor of 1% or greater, the fill factor being the ratio of the spacer contact area to the total plate area.
[Invention 1031]
The apparatus according to any one of the present invention, wherein a multiplication value of Young's modulus of the spacer and a filling rate of the spacer is 20 MPa or more, and the filling rate is a ratio of a spacer contact area to a total plate area.
[Invention 1032]
The device of any of the preceding claims, wherein the spacing between the two plates in the closed configuration is less than 200 μm.
[Invention 1033]
The device of any of the preceding inventions wherein the spacing between the two plates in the closed configuration is a value selected from 1.8 μm to 3.5 μm.
[Invention 1034]
The apparatus of any of the preceding claims, wherein the spacers are secured on the plate by directly embossing the plate or by injection molding into the plate.
[Invention 1035]
The apparatus of any of the preceding inventions wherein the material of the plate and the spacer is selected from polystyrene, PMMA, PC, COC, COP, or other type of plastic.
[Invention 1036]
The device according to any one of the preceding claims, wherein the spacer has a pillar shape, and a sidewall angle of the spacer has a circular shape having a radius of curvature of at least 1 µm.
[Invention 1037]
The spacer has a density of at least 1000/mm ² The device of any of the preceding claims, wherein:
[Invention 1038]
The device of any of the preceding inventions wherein at least one of the plates is transparent.
[Invention 1039]
The mold used to manufacture the spacers is manufactured by a mold including a plurality of features, the features being (a) directly, by reactive ion etching or ion beam etching, or (b) reactive. The apparatus of any of the preceding inventions made by replicating or multiple replicating features etched by ion etching or ion beam etching.
[Invention 1040]
(A) obtaining a blood sample,
(B) obtaining a first plate and a second plate moveable relative to each other in different configurations, each plate having a substantially flat sample contact surface Or two plates are flexible, one or two of said plates comprising spacers fixed to their respective sample contact surfaces, said spacers comprising:
i. A predetermined substantially uniform height,
ii. A pillar shape having a substantially uniform cross section and a flat top surface;
iii. Width to height ratio of 1 or more,
iv. A predetermined constant distance between the spacers in the range of 10 μm to 200 μm,
v. Filling rate of 1% or more,
vi. The product of the filling rate of the spacer and Young's modulus, which is 2 MPa or more,
With steps, as well as
(C) depositing the blood sample on one or two of the plates when the plates are configured in an open configuration, wherein the open configuration comprises the two plates partially or completely. A configuration in which the plates are spaced apart and the spacing between the plates is not adjusted by the spacers,
(D) after (c), using the two plates to compress at least a portion of the blood sample into a layer of substantially uniform thickness, The layer is confined by the sample contacting surface of the plate, the uniform thickness of the layer being controlled by the spacer and the plate and averaging within the range of 1.8 μm to 3 μm with a variation of less than 10%. Has a value, the compression is
Uniting the two plates, and
In order to press the plates together into the closed configuration, in parallel or in sequence, one or more regions of at least one of the plates are suitably pressed, and the appropriate said press is applied to the at least part of the sample. A substantially uniform pressure on the plate is generated such that the pressing causes the at least a portion of the sample to spread laterally between the sample contacting surfaces of the plate and the closed configuration to provide a uniform thickness region. A configuration in which the spacing between the plates in the layer is adjusted by the spacer, and
(E) analyzing blood in the layer of uniform thickness while the plate is in a closed configuration.
A method for analyzing a blood sample, comprising:
The filling factor is the ratio of the spacer contact area to the total plate area,
A suitable press is a method of making the pressure applied to a certain region substantially constant regardless of the shape change of the outer surface of the plate,
The method wherein the parallel press simultaneously applies pressure to a desired region and the sequential press applies pressure to a part of the desired region and gradually moves to another region.
[Invention 1041]
Removing external forces after the plate is in the closed configuration,
Imaging blood cells within the layer of uniform thickness while the plate is in the closed configuration; and
Counting the number of said blood cells within an area in the image
The method of the present invention 1040, comprising:
[Invention 1042]
The method according to the invention 1040 or 1041, wherein the distance between the spacers is in the range of 20 μm to 200 μm.
[Invention 1043]
The method according to any one of claims 1040 to 1042, wherein the distance between the spacers is in the range of 5 μm to 20 μm.
[Invention 1044]
The method of any of the invention 1040 through 1043 wherein the surface variation is less than 30 nm.
[Invention 1045]
The method according to any of 1040 to 1044 of the present invention, wherein the blood sample in (b) is undiluted whole blood to which an anticoagulant is not added.
[Invention 1046]
The step (b) of attaching is
i. Puncture human skin to expel a drop of blood onto the skin, ii. The method of any of claims 1040-104, wherein the method is performed by contacting the droplet of blood with one or two of the plates without the use of a blood transfer device.
[Invention 1047]
The method of any of 1040-1046, wherein said counting step (f) comprises counting the number of red blood cells.
[Invention 1048]
The counting step (f) includes the method of any of 1040 to 1047 of the present invention, which comprises counting the number of white blood cells.
[Invention 1049]
The present invention 1040, wherein said counting step (f) comprises staining cells in said sample and counting the number of neutrophils, lymphocytes, monocytes, eosinophils and basophils. ~ 1048 any method.
[Invention 1050]
The step of imaging and the step of counting,
i. Illuminating the blood cells within the layer of uniform thickness,
ii. Taking one or more images of the blood cells using a CCD or CMOS sensor,
iii. Identifying a cell in the image using a computer; and
iv. Counting the number of blood cells in the area of the image
The method according to any of claims 1040 to 1049 of the present invention, which is carried out by
[Invention 1051]
The method of any of claims 1040-1050, wherein the external force of step (c) is provided by a human hand.
[Invention 1052]
Further measuring the sodium, potassium, chloride, bicarbonate, blood urea, nitrogen, magnesium, creatinine, glucose, calcium, HDL cholesterol LDL cholesterol level, and/or triglyceride level in the layer of uniform thickness. The method of any one of the present invention 104-1051, which comprises.
[Invention 1053]
The method of any of the claims 1040-1052, further comprising a dry reagent applied on one or two plates.
[Invention 1054]
The method of any of claims 1040-1053, wherein the layer of uniform thickness sample has a thickness uniformity of up to ±5%.
[Invention 1055]
Any of the inventions 104-1054, wherein the spacer is a column having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof. Method.
[Invention 1056]
The method of any of the present invention 1040 through 1055, wherein the spacing between the spacers is approximately the average thickness of the RBC.
[Invention 1057]
The method of any of the inventions 1040-1056, wherein the analyzing step comprises the step of imaging cells in the blood.
[Invention 1058]
The method of any of 1040-157, wherein the cells comprise red blood cells, white blood cells, or platelets.
[Invention 1059]
The method of any of the invention 1040 to 1058, wherein the step of analyzing the blood comprises the step of imaging cancer cells, viruses, or bacteria in the blood.
[Invention 1060]
The method of any of 1040 to 1059 of the present invention, wherein the step of analyzing blood comprises detecting a protein or nucleic acid.
[Invention 1061]
The step of analyzing the blood includes measuring blood cells, the measuring the thickness of the sample using the spacer, determining the lateral area by imaging, and using a 2D image to measure red blood cells. The method of any of the present invention 1040 through 1060, comprising calculating an area.
[Invention 1062]
The method of any of the inventions 1040-61, wherein the step of analyzing the blood comprises measuring the concentration of red blood cells in the blood.
[Invention 1063]
The method of any one of the present invention 1040 to 1062, wherein the step of analyzing the blood includes the step of measuring the white blood cell concentration in the blood.
[Invention 1064]
The method of any of 1040 to 1063 of the present invention, wherein the step of analyzing the blood comprises the step of measuring the platelet concentration in the blood.
[Invention 1065]
First plate and second plate
Including,
v. The plates are movable relative to each other in different configurations,
vi. One or two of the plates is flexible,
vii. Each said plate has on its respective surface a sample contact area for contacting a blood sample,
viii. One or two of the plates includes spacers fixed to the respective plates, the spacers having a predetermined substantially uniform height and a predetermined constant inter-spacer distance within a range of 7 μm to 200 μm. And at least one of the spacers is in the sample contact area,
One configuration is an open configuration in which the two plates are separated and the spacing between the plates is not adjusted by the spacer and the sample adheres to one or two of the plates, and
Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein at least a portion of the sample is compressed by the two plates to a very uniform thickness. , The uniform thickness of said layers being limited by the inner surfaces of said two plates and controlled by said spacers and said plates, and with an average of within a range of 1.8 μm to 3 μm with small fluctuations. An apparatus for analyzing a liquid sample having a value.
[Invention 1066]
The apparatus of invention 1065, wherein the distance between the spacers is in the range of 14 μm to 200 μm.
[Invention 1067]
The device of the present invention 1065 or 1066, wherein the distance between the spacers is in the range of 7 μm to 20 μm.
[Invention 1068]
Any of the inventions 1065-1067, wherein the spacer is a column having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof. apparatus.
[Invention 1069]
Any of the inventions 1065-1068 wherein the spacer has a pillar shape and has a substantially flat upper surface, and for each spacer the ratio of its lateral dimension to the spacer height is at least 1. That device.
[Invention 1070]
The device according to any one of the present inventions 1065 to 1069, wherein the spacer has a pillar shape, and a sidewall angle of the spacer has a circular shape having a radius of curvature of at least 1 μm.
[Invention 1071]
The spacer has a density of at least 1000/mm ² The device of any of the inventions 1065-1070, which is
[Invention 1072]
The device of any of the present invention 1065-1071 wherein at least one of the plates is transparent.
[Invention 1073]
The device of any of the present invention 1065-1072 wherein at least one of said plates is made of a flexible polymer.
[Invention 1074]
The device of any of the present inventions 1065-1073, wherein the spacers are not compressible and/or independently only one of the plates is flexible.
[Invention 1075]
The device of any of the present invention 1065-1074 wherein the thickness of said flexible plate is in the range of 50 m to 200 m.
[Invention 1076]
The device of any of the present inventions 1065-1075, further comprising a dry reagent coated on one or two plates.
[Invention 1077]
The device of any of the present invention 1065-1076 wherein said variation is less than 10%.
[Invention 1078]
A method for locally binding a target entity in a portion of a liquid sample, the method comprising:
(A) obtaining said sample containing a target entity capable of diffusing into the sample,
(B) a step of obtaining a first plate and a second plate which are movable relative to each other in different configurations, one or two said plates being spacers fixed on each plate The spacer has a predetermined substantially uniform height, the first plate includes a binding site on its surface, the binding site has a predetermined region, and binds to the target entity. Fix it, step,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, wherein the closed configuration compresses at least a portion of the sample to a uniform thickness. A layer, said layer contacting the inner surfaces of said two plates, bounded by the inner surfaces and contacting said binding site, wherein the uniform thickness of said layer is adjusted by said spacer and said plate Smaller than 250 μm and substantially smaller than the linear dimension of the predetermined region of the binding site,
(E) After (d) and while the plate is in the closed configuration,
(1) incubating the sample for a relevant length of time and then stopping the incubation, or
(2) Incubating the sample for a time equal to or greater than the minimum value of the relevant time length, and then allowing the binding of the target entity to the binding site within a time equal to or less than the maximum value of the relevant time length. Steps to evaluate
And
The relevant length of time is
i. Greater than or equal to the time required for the target entity to diffuse through the thickness of the layer of uniform thickness in the closed configuration, and
ii. Significantly less than the time required for the target entity to diffuse laterally across the minimum lateral dimension of the binding site,
At the end of the incubation in (1) or during the evaluation in (2), most of the target entity bound to the binding site is from a relevant volume of the sample,
The incubation allows the target entity to bind to the binding site, and the relevant volume is a portion of the sample above the binding site in the closed configuration.
Including the method.
[Invention 1079]
Further comprising the step of assessing said target entity bound to said binding site, said assessing step being performed either after (i) step (e) or during (ii) step (e). The method of invention 1078, wherein the method is initiated after the sample has been incubated for a time that is approximately equal to or longer than the time required for the analyte to diffuse over the thickness of the uniform thickness layer.
[Invention 1080]
The method further comprises calculating the concentration of the analyte in the sample of the relevant volume, the calculation comprising the relevant sample volume defined by the predetermined region of the binding site, the uniform sample in the closed configuration. The method of the invention 1078 or 1079, which is based on the thickness of the sample and the evaluated target entity bound to the binding site.
[Invention 1081]
The method of any of claims 1078-1080, wherein said target entity is a capture agent, analyte, or detection agent.
[Invention 1082]
The method of any of claims 1078-1080, wherein the sample volume in the analyte assay region is approximately equal to the product of the predetermined area of the binding site and the predetermined spacer height.
[Invention 1083]
The first plate comprises on its surface a first predetermined assay site and a second predetermined assay site, the distance between the edges of the assay site being the uniform when the plate is in the closed position. Substantially greater than the thickness of a uniform thickness layer, at least a portion of the uniform thickness layer is above the predetermined assay site, and the sample is one or more capable of diffusing into the sample. The method of any of claims 1078-1082 of the present invention, which comprises an analyte of.
[Invention 1084]
The method of invention 1083, comprising assessing a first analyte at the first assay site and assaying a second analyte at the second predetermined assay site.
[Invention 1085]
The method of invention 1038, comprising assaying the same analyte at the first predetermined assay site and the second predetermined assay site.
[Invention 1086]
The first plate further has at least three analyte assay sites on its surface, and the distance between the edges of any two adjacent assay sites is such that the plate is in the uniform position when in the closed position. Substantially greater than the thickness of the layer of thickness, at least a portion of the layer of uniform thickness is above the assay site, and the sample is one or more analytes capable of diffusing into the sample. The method of any of 1078-1085 of the present invention, which comprises:
[Invention 1087]
The method of any of the inventions 1078-1086, comprising assaying a different analyte within each said assay site.
[Invention 1088]
Each said assay site further comprises a sub-analyte assay site, wherein at least two adjacent sub-analyte assay sites are separated by less than the thickness of said uniform thickness layer when said plate is in the closed position. Any of the inventions 1078-1087, wherein at least a portion of the layer of uniform thickness is above the assay site and the sample has one or more analytes capable of diffusing into the sample. the method of.
[Invention 1089]
The method of any of claims 1078-1088, wherein the analyte assay region is between a pair of electrodes.
[Invention 1090]
The method of any of claims 1078-1089 of the present invention, wherein said assay region is defined by one patch of dry reagent.
[Invention 1091]
The method of any of claims 1078-1090 of the invention, wherein said assay region binds and immobilizes said analyte.
[Invention 1092]
The method of any of claims 1078-1091 of the present invention, wherein said assay region amplifies the signal from said analyte.
[Invention 1093]
The assay region of claim 1078, wherein the assay region is defined by one patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. One of 1092 methods.
[Invention 1094]
The method of any of claims 1078-1093 of the present invention, wherein the thickness of said sample is less than 200 [mu]m.
[Invention 1095]
The method of any one of the present inventions 1078-1094, wherein when the plate is in the closed configuration, the first plate and the second plate include a plurality of storage sites opposite each other.
[Invention 1096]
The method of any of claims 1078-1095, wherein the ratio of the linear dimension of the binding site to the uniform thickness is greater than 5.
[Invention 1097]
The process of any of claims 1078-1096 wherein the reaction is saturated in less than 60 seconds.
[Invention 1098]
The method of any of claims 1078-1097, wherein said relevant time is in the range of 60 seconds to 30 minutes.
[Invention 1099]
In step e(i), the method of any of the present inventions 1078-1098, comprising assessing the target entity bound to the binding site after stopping the incubation.
[Invention 1100]
The method of any of claims 1078-1099 of the present invention comprising one or more washes.
[Invention 1101]
A device for locally binding a target entity on a portion of a liquid sample, comprising:
First plate and second plate
Including,
i. The plates are movable relative to each other in different configurations, one or two of the plates being flexible,
iii. Each said plate has a sample contact area on its respective surface for contacting a sample, said sample comprising an entity capable of diffusing into said sample,
iv. One of the plates has a binding site on its sample contact area, the binding site has a predetermined area, and binds to and immobilizes the target entity,
v. One or two of the plates includes spacers fixed to each plate, the spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers, and the spacers. At least one of which is in the sample contact area,
One configuration is an open configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacer and the sample adheres to one or two of the plates, And
Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein at least a portion of the sample is compressed by the two plates to form a layer of uniform thickness. And at least a portion of the layer of uniform thickness is above the bonding site, and the uniform thickness of the layer is limited by the inner surfaces of the two plates, the plate and the spacer A device of less than 250 μm and substantially less than the average linear dimension of a given region of the binding site.
[Invention 1102]
The first plate further comprises a first predetermined assay site and a second predetermined assay site on a surface thereof, the distance between the edges of the assay site being determined when the plate is in the closed position. Substantially greater than the thickness of the uniform thickness layer, at least a portion of the uniform thickness layer is above the predetermined assay site, and the sample is one capable of diffusing into the sample. The device of invention 1101 comprising the analytes described above.
[Invention 1103]
The first plate has at least three analyte assay sites on its surface, and the distance between the edges of any two adjacent assay sites is determined by the uniform thickness when the plate is in the closed position. Substantially greater than the thickness of the sample layer, at least a portion of the layer of uniform thickness is above the assay site and the sample contains one or more analytes that can diffuse into the sample. The apparatus of the present invention 1101 or 1102, having:
[Invention 1104]
The first plate has at least two adjacent analyte assay sites on its surface, wherein the analyte assay sites are less than the thickness of the uniform thickness layer when the plate is in the closed position. At least some of the layers of uniform thickness are not separated by a substantially large distance and are above the assay site, and the sample has one or more analytes capable of diffusing into the sample. The device of any of the present invention 1101-1103.
[Invention 1105]
The device of any of the present invention 1101-1104, wherein the analyte assay region is between a pair of electrodes.
[Invention 1106]
The device of any of the present invention 1101-1105, wherein the assay region is defined by one patch of dry reagent.
[Invention 1107]
The device of any of claims 1101-1106 of the present invention, wherein said assay region binds and immobilizes said analyte.
[Invention 1108]
The invention 1101 to 110, wherein the assay region is defined by one patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. Any device of 1107.
[Invention 1109]
The device of any of the present invention 1101-1108, wherein the distance between the spacers is in the range of 14 m to 200 m.
[Invention 1110]
The device of any of the present invention 1101-1109, wherein the distance between said spacers is in the range of 7-20 m.
[Invention 1111]
Any of the present invention 1101-1110, wherein the spacer is a column having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof. apparatus.
[Invention 1112]
Any of the inventions 1101-1111, wherein the spacer has a pillar shape and has a substantially flat upper surface, and for each spacer the ratio of its lateral dimension to the spacer height is at least 1. That device.
[Invention 1113]
The device according to any one of the present invention 1101 to 1112, wherein the spacer has a pillar shape, and a sidewall angle of the spacer has a circular shape having a radius of curvature of at least 1 μm.
[Invention 1114]
The spacer has a density of at least 1000/mm ² The device of any of the present invention 1101-1113.
[Invention 1115]
The device of any of the present invention 1101-1114 wherein at least one of said plates is transparent.
[Invention 1116]
The device of any of the present invention 1101-1115 wherein at least one of said plates is made of a flexible polymer.
[Invention 1117]
The device of any of the present invention 1101-1116 wherein only one of the plates is flexible.
[Invention 1118]
A method for locally releasing a reagent to a portion of a liquid sample, the method comprising:
(A) obtaining a sample,
(B) obtaining a first plate and a second plate moveable with respect to each other so as to have different configurations,
(I) one or two said plates include spacers fixed to each plate,
(Ii) the spacer has a predetermined uniform height, and
(Iii) the first plate comprises a storage site on its surface, said storage site having a predetermined region and containing a reagent, said reagent being dissolved in said sample upon contact with said sample, Diffusing into the sample, step,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration comprising the two plates being partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, wherein the closed configuration compresses at least a portion of the sample to a uniform thickness. A layer, said layer being constrained by the inner surfaces of said two plates and covering said storage site, wherein the uniform thickness of said layer is adjusted by said spacer and said plate and is less than 250 μm And substantially smaller than the linear dimension of the predetermined area of the storage site,
(E) after (d) and while the plate is in the closed configuration, incubating the sample for a relevant length of time and then stopping the incubation,
The relevant length of time is
i. A time that is approximately equal to or longer than the time required for the target entity to diffuse through the thickness of the layer of uniform thickness in the closed configuration, and
ii. Less than the time required for the target entity to laterally diffuse over the linear dimension of a given region of the binding site,
Thereby, after incubation, the majority of the reagents initially on the storage site are in the relevant volume of the sample,
The incubation is a process that allows the reagents to bind or mix with the sample, the relevant volume being the portion of the sample above the binding site in the closed configuration.
Including the method.
[Invention 1119]
The method of invention 1118 wherein the reagent is a capture agent, an analyte, or a detection agent.
[Invention 1120]
The surface of the second plate in step (b) further comprises a binding site, said binding site having a corresponding predetermined region and a corresponding position and binding to said reagent to immobilize it,
In step (d), the binding site and the reagent site are aligned in the closed configuration, and
At the end of the incubation of step (e), the majority of the reagent initially on the storage site is bound to the binding site, and the binding site and the reagent in the relevant volume are substantially bound. 1118 or 1119 of the present invention, wherein equilibrium is reached.
[Invention 1121]
The method further comprises calculating the concentration of the analyte in the sample of the relevant volume, the calculation comprising the relevant sample volume defined by a predetermined region of the storage site, the uniform sample in the closed configuration. The method of any of the inventions 1118-1120, which is based on the thickness of the sample and the amount of target entity detected.
[Invention 1122]
The method of any of the present invention 1118-1121, wherein said target entity is a capture agent, analyte, or detection agent.
[Invention 1123]
The method of any of the inventions 1118-1122, wherein the volume of the sample in the predetermined region is approximately equal to the product of the predetermined area and the predetermined spacer height.
[Invention 1124]
The first plate includes on its surface a first storage site and a second storage site, the distance between the edges of the storage site being equal to the uniform thickness when the plate is in the closed position. Substantially greater than the thickness of the layer, at least a portion of the layer of uniform thickness is above the storage site, and the sample has one or more analytes capable of diffusing into the sample, The method of any of claims 1118-1123 of the present invention.
[Invention 1125]
The method of invention 1124, comprising assessing a first analyte on said first storage site and assaying a second analyte on said second storage site.
[Invention 1126]
The method of invention 1124, comprising assaying the same analyte on said first storage site and on said second storage site.
[Invention 1127]
The first plate further comprises at least three storage sites on its surface, the distance between the edges of any two adjacent storage sites being equal to the uniform thickness when the plate is in the closed position. Substantially greater than the thickness of the layer, at least a portion of the layer of uniform thickness is above the predetermined region, and the sample has one or more analytes capable of diffusing into the sample. The method of any of the inventions 1118-1126.
[Invention 1128]
The method of invention 1127, comprising assaying a different analyte within each said storage site.
[Invention 1129]
The method of any of claims 1118-1128, wherein the storage site is defined by one patch of dry reagent.
[Invention 1130]
1118-wherein the storage site is defined by one patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. 1129 either way.
[Invention 1131]
The method of any of claims 1118-1130 of the present invention, wherein the thickness of said sample is less than 200 [mu]m.
[Invention 1132]
The method of any of the present invention 1118-1131, wherein the first plate and the second plate include storage sites in opposing positions when the plate is in the closed configuration.
[Invention 1133]
The method of any of claims 1118-1132, wherein the ratio of the linear dimension of the binding site to the uniform thickness is greater than 5.
[Invention 1134]
The method of any of claims 1118-1133 wherein the reaction is saturated in less than 60 seconds.
[Invention 1135]
The method of any of claims 1118-1134, wherein said relevant time is in the range of 60 seconds to 30 minutes.
[Invention 1136]
The method of any of the present invention 1118-1135, which comprises one or more washes.
[Invention 1137]
First plate and second plate
Including,
i. The plates are movable with respect to each other in different configurations,
ii. One or two of the plates is flexible,
vi. Each said plate has a sample contact area on its respective surface for contacting a sample,
vii. One of the plates includes a storage site on its sample contact area, the storage site having a predetermined area and containing a reagent, wherein the reagent dissolves in the sample upon contact with the sample. Diffuse into the sample and bind to the target entity,
viii. One or two of the plates include spacers fixed to the respective plates, the spacers being (a) 250 μm or less and substantially smaller than the average linear dimension of the predetermined area of the reagent site. A substantially uniform height of, and (b) a predetermined constant distance between the spacers of 200 μm or less, at least one spacer being within the sample contact region,
One configuration is an open configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacer and the sample adheres to one or two of the plates, And
Another configuration is a closed configuration that is configured after the sample is deposited in an open configuration, in which at least a portion of the sample is compressed by the two plates into a layer of uniform thickness. At least a portion of the layer of uniform thickness is above the bonding site, and the uniform thickness of the layer is limited by the inner surfaces of the two plates and by the spacer and the plate. A device for locally releasing a reagent to a portion of a liquid sample that is conditioned.
[Invention 1138]
The first plate further includes, on its surface, a second storage site having a predetermined area, and a distance between edges of the first storage site and the second storage site is a closed position of the plate. Substantially greater than the thickness of the uniform thickness layer when at, at least a portion of the uniform thickness layer is above the storage site and the sample diffuses into the sample. The device of the present invention 1137 having one or more analytes capable.
[Invention 1139]
The first plate has at least three storage sites on its surface and the distance between the edges of any two adjacent storage sites is equal to the uniform thickness when the plate is in the closed position. Substantially greater than the thickness of the layer, at least a portion of the layer of uniform thickness is above the storage site and the sample has one or more analytes capable of diffusing into the sample, The device of the present invention 1137 or 1138.
[Invention 1140]
The first plate has at least two adjacent storage sites on its surface, the storage sites having a distance substantially greater than the thickness of a layer of uniform thickness when the plate is in the closed position. 1137-, wherein at least some of the uniform thickness layers are above assay sites and the sample has one or more analytes that can diffuse into the sample. Any device of 1139.
[Invention 1141]
The device of any of the present invention 1137-1140 wherein the storage site is defined by one patch of dry reagent.
[Invention 1142]
1137-wherein the storage site is defined by one patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. Any of the 1141 devices.
[Invention 1143]
The device of any of the present invention 1137-1142, wherein the distance between the spacers is in the range of 14 m-200 m.
[Invention 1144]
The device of any of the present invention 1137-1143, wherein the distance between the spacers is in the range of 7 μm to 20 μm.
[Invention 1145]
The spacer is columnar having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof. apparatus.
[Invention 1146]
Any of the inventions 1137-1145, wherein the spacer has a pillar shape and has a substantially flat upper surface, and for each spacer the ratio of its lateral dimension to the spacer height is at least 1. That device.
[Invention 1147]
The device of any of the present invention 1137-1146, wherein the spacer has a pillar shape and the sidewall angle of the spacer has a circular shape with a radius of curvature of at least 1 μm.
[Invention 1148]
The spacer has a density of at least 1000/mm ² The device of any of claims 1137-1147 of the present invention.
[Invention 1149]
The device of any of the present invention 1137-1148 wherein at least one of the plates is transparent.
[Invention 1150]
The device of any of the present invention 1137-1149 wherein at least one of the plates is made of a flexible polymer.
[Invention 1151]
The device of any of the present invention 1137-1150 wherein only one of the plates is flexible.
[Invention 1152]
A method for reducing the time to bind a target entity in a relevant volume of a sample onto a binding site on a plate surface, the method comprising:
(A) obtaining a sample containing a target entity capable of diffusing into the sample,
(B) Obtaining a first plate and a second plate that are movable relative to each other in different configurations, wherein one or two said plates are spacers fixed on their respective plates. The one or two plates are flexible, the spacers have a predetermined substantially uniform height, and a predetermined constant distance between the spacers, and the first plate has a surface thereof. A binding site, wherein the binding site has a predetermined region, and binds to and immobilizes the target entity.
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, wherein the closed configuration has an associated volume of the sample thickness of the plate open. A layer having a thickness that is substantially uniform compared to the thickness of the layer, the layer having a thickness of at least 1 mm. ² A layer of substantially uniform thickness, the layer of substantially uniform thickness being limited by the inner surfaces of the two plates and covering the binding site, the uniform thickness of the layer being Adjusted by said plate and smaller than 250 μm and substantially smaller than the linear dimension of said predetermined region of said binding site, said relevant volume being part or all of the volume of said sample,
Including,
The method, wherein reducing the thickness of the sample in the relevant volume reduces the time for the binding between the binding site and the target entity in the relevant volume to reach equilibrium.
[Invention 1153]
The method of invention 1152, comprising reducing at least a portion of the thickness of the sample.
[Invention 1154]
The predetermined area is at least 1 mm ² The method of the invention 1152 or 1153, which is
[Invention 1155]
The predetermined area is 1 mm ² The method of any of 1152-1154, wherein the method is less than.
[Invention 1156]
The method further comprises assessing the target entity bound to the binding site, wherein the assessing step is approximately equal to or the time required for the analyte to diffuse over the thickness of the layer of uniform thickness. The method of any of claims 1152-1155, wherein the method is initiated after the sample has been incubated for a longer period of time.
[Invention 1157]
The method of any of the invention 1152-1156, wherein said time is reduced to less than 60 seconds.
[Invention 1158]
The method further comprises calculating the concentration of the analyte in the sample of the relevant volume, the calculation comprising the relevant sample volume defined by the predetermined region of the binding site, the uniform sample in the closed configuration. The method of any of claims 1118-1157 of the present invention, based on the thickness of the sample and the evaluated target entity bound to said binding site.
[Invention 1159]
The method of any of claims 1152-1158, wherein the target entity is a capture agent, analyte, or detection agent.
[Invention 1160]
The method of any of claims 1152-1159, wherein the sample volume at the analyte binding site is approximately equal to the product of the predetermined area of the binding site and the predetermined spacer height.
[Invention 1161]
The first plate comprises on its surface a first binding site and a second binding site, the distance between the edges of the binding site being equal to the uniform thickness of the plate when in the closed position. Substantially greater than the thickness of the layer, at least a portion of the layer of uniform thickness is above the binding site, and the sample has one or more analytes capable of diffusing into the sample, The method of any of 1152-1160 of the present invention.
[Invention 1162]
The method of any of claims 1152-1116, which comprises the step of assessing a first analyte at said first binding site and assaying a second analyte at said second binding site.
[Invention 1163]
115. The method of any of the invention 1152-1162, comprising assaying the same analyte at the first binding site and the second binding site.
[Invention 1164]
The first plate further has at least three analyte binding sites on its surface, and the distance between the edges of any two adjacent binding sites is such that the plate is in the uniform position when in the closed position. Substantially greater than the thickness of the layer of thickness, at least a portion of the layer of uniform thickness is above the binding site, and the sample is one or more analytes capable of diffusing into the sample. The method of any of 1152-1163, wherein the method comprises:
[Invention 1165]
The method of invention 1164, comprising assaying a different analyte within each said binding site.
[Invention 1166]
Each said binding site further comprises a sub-analyte binding site, wherein at least two adjacent sub-analyte binding sites are separated by less than the thickness of said uniform thickness layer when said plate is in the closed position. 1164, wherein at least a portion of the layer of uniform thickness is above the binding site and the sample has one or more analytes that can diffuse into the sample.
[Invention 1167]
The method of any of 1152-1166, wherein the binding site is between a pair of electrodes.
[Invention 1168]
The method of any of claims 1152-1167, wherein the binding site is defined by one patch of dry reagent.
[Invention 1169]
The method of any of claims 1152-1168, wherein the binding site binds to and immobilizes the analyte.
[Invention 1170]
The method of any of claims 1152-1169, wherein said binding site amplifies the signal from said analyte.
[Invention 1171]
The invention 1152, wherein the binding site is defined by one patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. ~ 1170 one of the ways.
[Invention 1172]
The method of any of claims 1152-1171, wherein the thickness of the sample is less than 200 μm.
[Invention 1173]
The method of any of claims 1152-1172, wherein the first plate and the second plate include a plurality of storage sites in opposing positions when the plate is in the closed configuration.
[Invention 1174]
The method of any of claims 1152-1173, wherein the ratio of the linear dimension of the binding site to the uniform thickness is greater than 5.
[Invention 1175]
The method of any of claims 1152-1174 wherein the reaction is saturated in less than 60 seconds.
[Invention 1176]
The method of any of claims 1152-1175, wherein said relevant time is in the range of 60 seconds to 30 minutes.
[Invention 1177]
In step e(i), the method of any of claims 1152-1176, comprising assessing the target entity bound to the binding site after stopping the incubation.
[Invention 1178]
The method of any of 1152-1177 of the present invention, comprising one or more washes.
[Invention 1179]
First plate and second plate
Including,
i. The plates are movable relative to each other in different configurations, one or two plates being flexible,
iii. Each said plate has a sample contact area on its respective surface for contacting a sample, said sample comprising an entity capable of diffusing into said sample,
iv. One of the plates has a binding site on its sample contact area, the binding site has a predetermined area, and binds to and immobilizes a target entity,
v. One or two of the plates include spacers secured to the respective plates, the spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers, and the spacers. At least one of which is in the sample contact area,
One configuration is an open configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacer and the sample adheres to the one or two plates, And
Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein at least a portion of the sample is compressed by the two plates to form a layer of uniform thickness. At least a portion of the layer of uniform thickness is above the bonding site, and the uniform thickness of the layer is limited by the inner surfaces of the two plates, the plate and the spacer Smaller than 250 μm and substantially smaller than the average linear dimension of the predetermined region of said binding site, and
By reducing the thickness of the relevant volume of the sample, the time for the binding between the binding site and the target entity in the relevant volume to reach equilibrium is shortened. A device for locally binding the target entity in part.
[Invention 1180]
The first plate comprises on its surface a first binding site and a second binding site, the distance between the edges of the binding site being equal to the uniform thickness of the plate when in the closed position. Substantially greater than the thickness of a layer, at least a portion of the layer of uniform thickness is above the binding site, and the sample has one or more analytes that can diffuse into the sample, The device of the invention 1179.
[Invention 1181]
The first plate has at least three analyte binding sites on its surface and the distance between the edges of any two adjacent binding sites is determined by the uniform thickness when the plate is in the closed position. Substantially greater than the thickness of the layer, at least a portion of the layer of uniform thickness is above the binding site and the sample contains one or more analytes that can diffuse into the sample. The apparatus of the present invention 1179.
[Invention 1182]
The first plate has at least two adjacent analyte binding sites on its surface, the analyte binding sites being substantially greater than the thickness of a layer of uniform thickness when the plate is in the closed position. At least part of the uniform thickness layer is above the binding site and the sample has one or more analytes capable of diffusing into the sample. The apparatus of the present invention 1179.
[Invention 1183]
The device of any of the present invention 1179-1182 wherein the binding site is defined by one patch of dry reagent.
[Invention 1184]
The device of any of claims 1179-1183 of the present invention, wherein said binding site binds and immobilizes said analyte.
[Invention 1185]
1179-wherein the binding site is defined by a patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. Any of the devices in 1184.
[Invention 1186]
The device of any of the present invention 1179-1185, wherein the distance between the spacers is in the range of 14 μm to 200 μm.
[Invention 1187]
The device of any of the present invention 1179-1186, wherein the distance between the spacers is in the range of 7 μm to 20 μm.
[Invention 1188]
The spacer is columnar having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof, any of the inventions 1179-1187. apparatus.
[Invention 1189]
Any of the inventions 1179-1188, wherein the spacer has a pillar shape and has a substantially flat upper surface, and for each spacer the ratio of its lateral dimension to the height of the spacer is at least 1. That device.
[Invention 1190]
The device of any of the present invention 1179-1189, wherein the spacer has a pillar shape and the sidewall angle of the spacer has a circular shape with a radius of curvature of at least 1 μm.
[Invention 1191]
The spacer has a density of at least 1000/mm ² The device of any of claims 1179-1190 of the present invention.
[Invention 1192]
The device of any of the present invention 1179-1191 wherein at least one of the plates is transparent.
[Invention 1193]
The device of any of the present invention 1179-1192 wherein at least one of the plates is made of a flexible polymer.
[Invention 1194]
The device of any of the present invention 1179-1193 wherein only one of the plates is flexible.
[Invention 1195]
A method for parallel multiplexed assay of a liquid sample without fluidic isolation, comprising:
(A) obtaining said sample containing one or more target analytes capable of diffusing into the sample,
(B) obtaining a first plate and a second plate moveable with respect to each other so as to have different configurations,
i. One or two said plates comprising spacers fixed to the respective plates, one or two plates being flexible,
ii. The spacers have a predetermined substantially uniform height and a predetermined constant distance between the spacers,
iii. The first plate has one or more binding sites on its surface, each said binding site comprising a predetermined region containing a capture agent that binds and immobilizes the corresponding target analyte of (a). And has
iv. The second plate has one or more corresponding storage sites on its surface, each storage site having a predetermined region and containing a concentration of a detection agent, the detection agent being in the sample. Upon contact, dissolves in the sample and diffuses into the sample,
Each capture agent, target analyte, and corresponding detection agent can form a capture agent-target analyte-detection agent sandwich at the binding site of the first plate,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration comprising the two plates being partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, the closed configuration comprising:
i. At least a portion of the sample is compressed into a layer of uniform thickness, the layer of uniform thickness contacting and limited by the inner surfaces of the two plates, and one or more Contacting said binding site and one or more said storage sites of
ii. One or more corresponding said storage sites are on one or more said binding sites, and
iii. A uniform thickness of the layer, adjusted by the spacer and the plate, is less than 250 μm and substantially less than the linear dimension of a given area of each storage site.
Is a step,
(E) After (d) and while the plate is in the closed configuration,
(1) incubating the sample for a relevant length of time and then stopping the incubation, or
(2) Incubating the sample for a time greater than or equal to the minimum relevant time length and then binding each target analyte to the binding site within a time less than or equal to the maximum relevant time length. The steps to evaluate
And
The relevant length of time is
i. More than the time required for the target analyte of (a) to diffuse over the thickness of the layer of uniform thickness in the closed configuration, and
ii. Significantly shorter than the time required for the target analyte of (a) to diffuse laterally over the smallest linear dimension of a given region of the storage or binding site,
Thereby, a reaction takes place, in which in the end of the incubation in (1) or during the evaluation in (2) most of the capture agent-target analyte-detection agent sandwich bound to each binding site has a corresponding Derived from the relevant volume of the sample,
The incubation allows each target analyte to bind to a binding site and a detection agent and the corresponding relevant volume is the portion of the sample above the corresponding storage site in the closed configuration. The separation distance between the edges of the adjacent storage sites and the separation distance between the edges of the adjacent binding sites is greater than the distance over which the target analyte or detection agent can diffuse within the relevant time period, and There is no fluidic separation between the binding site and/or the storage site, a step
Including the method.
[Invention 1196]
The method of the present invention 1195, wherein the first plate has a plurality of the binding sites on its surface.
[Invention 1197]
The method of invention 1196, wherein each of the plurality of binding sites binds to a different target analyte.
[Invention 1198]
The method of any of the inventions 1195-1197, wherein said second plate has a plurality of said corresponding storage sites on its surface.
[Invention 1199]
The method of any of the inventions 1195-1198, wherein each of said plurality of corresponding storage sites binds to a different target analyte.
[Invention 1200]
The first plate has a plurality of the binding sites on its surface and the second plate has a plurality of the corresponding storage sites on its surface, each of which when the plate is in the closed configuration. The method of any of the present invention 1195-1199, wherein the binding site opposes the corresponding storage site.
[Invention 1201]
The first plate has a plurality of the binding sites on its surface and the second plate has a storage site on its surface, the binding sites of the binding sites when the plate is in the closed configuration. The method of any of claims 1195-1200, wherein at least some of the binding sites oppose a region within said storage site.
[Invention 1202]
The first plate has one binding site on its surface, the second plate has a plurality of storage sites on its surface, and at least some of the aforementioned when the plate is in the closed configuration. The method of any of the present invention 1195-1201, wherein the storage site opposes a region within said binding site.
[Invention 1203]
Any of the inventions 1195-1202, wherein the first plate has a plurality of binding sites on its surface, the binding sites comprising different capture agents that bind and immobilize the same target analyte. the method of.
[Invention 1204]
The method of any of the inventions 1195-1203, wherein the first plate has a plurality of binding sites on its surface, the binding sites comprising the same capture agent.
[Invention 1205]
The method of invention 1204, wherein the capture agent is present at different densities at the different binding sites.
[Invention 1206]
Any of the inventions 1195-1205, wherein there is a separation distance between two adjacent binding sites or two adjacent storage sites and the ratio of separation distance to thickness of the sample in the closed configuration is at least 3. That way.
[Invention 1207]
The method further comprises calculating a concentration of one or more analytes in the sample of the relevant volume, the calculation comprising the relevant sample volume defined by a predetermined region, the uniform sample in the closed configuration. The method of any of the inventions 1195-1206, which is based on sample thickness and the amount of target entity detected.
[Invention 1208]
The method of any of the inventions 1195-1207, wherein said target entity is a capture agent, analyte, or detection agent.
[Invention 1209]
The method of any of claims 1195-1208, wherein the volume of the sample in the predetermined region is approximately equal to the product of the predetermined area and the predetermined spacer height.
[Invention 1210]
The method of any of the inventions 1195-1209, comprising assaying the same analyte in the relevant volume on at least two of the binding sites.
[Invention 1211]
The method of any of the inventions 1195-1210, comprising assaying different analytes in the relevant volume on at least two of the binding sites.
[Invention 1212]
The method of any of the present invention 1195-1211, wherein said storage site is defined by a plurality of patches of dry reagent.
[Invention 1213]
The reservoir site is defined by a patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. Either way.
[Invention 1214]
The method of any of claims 1195-1213 of the present invention wherein the thickness of said sample is less than 200 [mu]m.
[Invention 1215]
The method of any of the present invention 1195-1214 wherein the ratio of the linear dimension of the binding site to the uniform thickness is greater than 5.
[Invention 1216]
The method of any of the present invention 1195-1215 comprising one or more washes.
[Invention 1217]
An apparatus for parallel multiplexed assay of a liquid sample without fluidic isolation, comprising:
Including a first plate and a second plate,
i. The plates are movable relative to each other in different configurations, one or two plates being flexible,
ii. One or two said plates comprising spacers fixed to the respective plates, said spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers,
iii. Each said plate has a sample contact area on its respective surface for contacting a sample, said sample comprising one or more target analytes capable of diffusing into said sample,
iv. The first plate has on its surface one or more binding sites each having a predetermined region containing a capture agent that binds and immobilizes a corresponding target analyte of the sample, and
v. The second plate has one or more corresponding storage sites on its surface, each storage site having a predetermined area and containing a concentration of a detection agent, the detection agent comprising: Upon contact with the sample, it dissolves in the sample and diffuses into the sample,
Each capture agent, target analyte, and corresponding detection agent can form a capture agent-target analyte-detection agent sandwich at the binding site of the first plate,
One configuration is an open configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by the spacer and the sample adheres to one or two of the plates, And
Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein the closed configuration comprises:
i. At least a portion of the sample is compressed into a layer of uniform thickness, the layer of uniform thickness contacting and limited by the inner surfaces of the two plates, and one or more Coating the binding site and one or more of the storage sites,
ii. One or more corresponding said storage sites are on one or more said binding sites, and
iii. A uniform thickness of the layer, adjusted by the spacer and the plate, is less than 250 μm and substantially less than the linear dimension of the predetermined area of each storage site, and
iv. There is no fluid isolation between the binding site and/or the storage site,
The separation between the edges of adjacent storage sites and the separation between the edges of adjacent binding sites is greater than the distance over which the target analyte or detection agent can diffuse within a relevant time period, and /Or a device in which there is no fluidic isolation between the storage sites.
[Invention 1218]
The device of invention 1217, wherein said first plate has a plurality of said binding sites on its surface.
[Invention 1219]
The device of invention 1217 or 1218, wherein each of the plurality of binding sites binds to a different target analyte.
[Invention 1220]
The device of any of claims 1217-1219, wherein the second plate has a plurality of corresponding storage sites on a surface thereof.
[Invention 1221]
The device of any of claims 1217-1220, wherein each of the plurality of corresponding storage sites binds a different target analyte.
[Invention 1222]
The first plate has a plurality of the binding sites on its surface and the second plate has a plurality of the corresponding storage sites on its surface, each of which when the plate is in the closed configuration. The device of any of the present inventions 1217-1221, wherein the binding site faces the corresponding storage site.
[Invention 1223]
The first plate has a plurality of the binding sites on its surface and the second plate has a storage site on its surface, the binding sites of the binding sites when the plate is in the closed configuration. The device of any of the present invention 1217-1222 wherein at least some of the binding sites oppose an area within said storage site.
[Invention 1224]
The first plate has one binding site on its surface and the second plate has multiple storage sites on its surface, among the storage sites when the plate is in the closed configuration. The device of any of the inventions 1217-1223, wherein at least some of the storage sites of said are facing an area within said binding site.
[Invention 1225]
Any of the inventions 1217-1224, wherein the first plate has a plurality of binding sites on its surface, the binding sites comprising different capture agents that bind to and immobilize the same target analyte. Equipment.
[Invention 1226]
The device of any of the present invention 1217-1225, wherein said first plate has a plurality of binding sites on its surface, said binding sites comprising the same capture agent.
[Invention 1227]
The device of invention 1226 wherein the capture agents are present at different densities at different binding sites.
[Invention 1228]
Any of the inventions 1217-1227 wherein there is a separation distance between two adjacent binding sites or two adjacent storage sites and the ratio of the separation distance to the thickness of the sample in the closed configuration is at least 3. That device.
[Invention 1229]
The device of any of claims 1217-1228, wherein the distance between the spacers is in the range of 1 μm to 120 μm.
[Invention 1230]
The device of any of claims 1217-1229, wherein the flexible plate has a thickness in the range of 20 m to 250 m and a Young's modulus in the range of 0.1 GPa to 5 GPa.
[Invention 1231]
An apparatus according to any of the inventions 1217-1230, wherein for the flexible plate, the product of the thickness of the flexible plate and the Young's modulus of the flexible plate is in the range of 60-750 GPa-μm. ..
[Invention 1232]
The layer of the sample of uniform thickness is at least 1 mm ² The device of any of the inventions 1217-1231 which is uniform over the lateral area of the.
[Invention 1233]
The apparatus of any of the present inventions 1217-1232, wherein the layer of uniform thickness sample has a thickness uniformity of up to ±5%.
[Invention 1234]
Any of the inventions 1217-1233, wherein the spacer is a pillar having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof. apparatus.
[Invention 1235]
The spacers have a pillar shape, have a substantially flat upper surface, and have a substantially uniform cross-section, and for each spacer, the ratio of its lateral dimension to the height of the spacer is at least 1. The apparatus of any of the present invention 1217-1234.
[Invention 1236]
The device of any of the present invention 1217-1235 wherein the distance between the spacers is periodic.
[Invention 1237]
The apparatus of any of the present invention 1217-1236, wherein the spacer has a fill factor of 1% or greater, the fill factor being the ratio of the spacer contact area to the total plate area.
[Invention 1238]
The apparatus according to any one of the present inventions 1217 to 1237, wherein a multiplication value of Young's modulus of the spacer and a filling rate of the spacer is 20 MPa or more, and the filling rate is a ratio of a spacer contact area to a total plate area.
[Invention 1239]
The device of any of the present invention 1217-1238, wherein the spacing between the two plates in the closed configuration is less than 200 μm.
[Invention 1240]
The device of any of the present invention 1217-1239, wherein the distance between the two plates in the closed configuration is a value selected from 1.8 μm to 3.5 μm.
[Invention 1241]
The device of any of the inventions 1217-1240, wherein the spacers are secured on the plate by directly embossing the plate or by injection molding into the plate.
[Invention 1242]
The device of any of the present invention 1217-1241, wherein the material of the plates and spacers is selected from polystyrene, PMMA, PC, COC, COP, or other type of plastic.
[Invention 1243]
The device of any of the inventions 1217-1242, wherein the spacer has a pillar shape and the sidewall angle of the spacer has a circular shape with a radius of curvature of at least 1 μm.
[Invention 1244]
The spacer has a density of at least 1000/mm ² The device of any of the present invention 1217-1243.
[Invention 1245]
The device of any of the present invention 1217-1244 wherein at least one of said plates is transparent.
[Invention 1246]
A system for rapidly analyzing a sample using a mobile phone,
(A) one or two plates of the CROF device are movable with respect to each other in different configurations,
i. One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of the plates, and
ii. Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein at least a portion of the sample is compressed by the two plates to form a layer of uniform thickness. A layer of uniform thickness is in contact with and limited by the inner surfaces of the two plates and is regulated by the plates and the spacer, a CROF device;
(B) i. One or more cameras for detecting and/or imaging the sample,
ii. Electronic components, signal processor, hardware and software for receiving and/or processing detected signals and/or images of said sample and for telecommunications
A mobile communication device including
(C) a light source from either the mobile communication device or an external source
Including the system.
[Invention 1247]
One of the plates has a binding site for binding an analyte, at least a portion of the layer of uniform sample thickness is above the binding site, and is greater than the average lateral linear dimension of the binding site. The system of the invention 1246, which is also substantially smaller.
[Invention 1248]
(D) A housing configured to hold the sample and be attached to the mobile communication device
The system of invention 1246 or 1247, further comprising:
[Invention 1249]
The housing comprises optics for facilitating imaging and/or signal processing of the sample by the mobile communication device, and a fixture configured to hold the optics to the mobile communication device. The system of the invention 1248, including:
[Invention 1250]
The mobile communication device of any of the inventions 1246-1249, wherein the mobile communication device is configured to communicate test results to a medical professional, medical institution, or insurance company.
[Invention 1251]
The system of invention 1250, wherein the mobile communication device is further configured to communicate information about the subject to a medical professional, medical institution, or insurance company.
[Invention 1252]
The system of any of the inventions 1246-1251, wherein the mobile communication device is configured to receive prescriptions, diagnoses, or advice from a medical professional.
[Invention 1253]
The mobile communication device is
(A) for capturing an image of the sample,
(B) for analyzing inspection and control positions in the image, and
(C) for comparing the value obtained from the analysis of the inspection position with a threshold characterizing a rapid diagnostic test
The system according to any one of the inventions 1246 to 1252, which is composed of hardware and software.
[Invention 1254]
The system of any of claims 1246-1253 wherein at least one of the plates comprises a storage site in which an assay reagent is stored.
[Invention 1255]
The system of any of claims 1246-1254 wherein at least one of the cameras reads a signal from the CROF device.
[Invention 1256]
The system of any of claims 1246 to 1255, wherein the mobile communication device communicates with a remote location via WiFi or a cellular network.
[Invention 1257]
The system of any of claims 1246 to 1255, wherein the mobile communication device is a mobile phone.
[Invention 1258]
A method for rapidly analyzing a sample using a mobile phone, comprising:
(A) depositing a sample on the CROF device of the system of any of the inventions 1246 to 1257,
(B) assaying the sample deposited on the CROF device to produce a result; and
(C) transmitting the result from the mobile communication device to a location remote from the mobile communication device.
Including the method.
[Invention 1259]
Analyzing the results at a remote location and providing analysis results; and
Transmitting the analysis result from the remote location to the mobile communication device
The method of the invention 1258, comprising:
[Invention 1260]
The method of claim 1258 or 1259, wherein said analysis is performed by a remote medical professional.
[Invention 1261]
The method of any one of the inventions 1258-1260, wherein the mobile communication device receives prescriptions, diagnostics, or advice from a remote medical professional.
[Invention 1262]
The method of any of the present invention 1259-1261, wherein said sample is a body fluid.
[Invention 1263]
The method according to any one of the inventions 1259 to 1262, wherein the sample is blood, saliva, or urine.
[Invention 1264]
The method of any of the inventions 1259-1263, wherein the assaying step comprises detecting an analyte in the sample.
[Invention 1265]
The method of invention 1264 wherein the analyte is a biomarker.
[Invention 1266]
The method of invention 1264, wherein the analyte is a protein, nucleic acid, cell, or metabolite.
[Invention 1267]
The method of any of the invention 1259-1266, comprising the step of counting the number of red blood cells.
[Invention 1268]
The method of any of claims 1259 to 1267 of the present invention comprising counting the number of white blood cells.
[Invention 1269]
The method of any of the present inventions 1259 to 1268, which comprises the step of staining cells in said sample and counting the number of neutrophils, lymphocytes, monocytes, eosinophils and basophils.
[Invention 1270]
The method of any of claims 1259-1269 of the invention wherein the assay performed in step (b) is a binding assay or a biochemical assay.
[Invention 1271]
(A) obtaining said sample containing an analyte capable of diffusing into the sample,
(B) obtaining a first plate and a second plate that are movable relative to each other in different configurations, wherein one or two of the plates comprises spacers fixed to the respective plates. Wherein the spacer has a predetermined uniform height and the first plate includes an analyte assay region having a predetermined region on its surface,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration comprising the two plates being partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) after (c), compressing at least a portion of the sample into a layer of uniform thickness using the two plates, wherein the layer of uniform thickness comprises: Confined by the inner surfaces of the two plates, at least a portion of the layer is above the analyte assay region, and the uniform thickness of the layer is controlled by the spacer and the plate and the analyte Substantially smaller than the linear dimension of a given lateral area of the assay area, said compression is
Uniting the two plates, and
Applying an external force to the outer surface of the plate to press the plate together into a closed configuration
The force creates a pressure on the plate over at least a portion of the sample, the pressure spreading at least a portion of the sample laterally between the inner surfaces of the plate, and The closed configuration is a configuration in which the spacing between the plates in the layer of uniform thickness region is adjusted by the spacer,
(E) while the plate is in the closed configuration, (i) for a time substantially equal to or longer than the time required for the analyte to diffuse over the thickness of the layer of uniform thickness, and (I) incubating the sample for a time significantly shorter than the time required for the analyte to diffuse across the analyte assay region,
(F) Immediately after step (e), the incubation is stopped and the analyte in the assay area is counted, or the incubation is continued while the plate is in the closed configuration, whereby the analyte is analyzed. Measuring the analyte in the assay area in a time significantly shorter than the time required to diffuse across the assay area.
A method for analyzing a liquid sample, comprising:
[Invention 1272]
The method of invention 1271, further comprising the step of: (g) calculating the analyte concentration in the volume of the sample in the sample layer in said assay region.
[Invention 1273]
The method of the invention 1271 or 1272, wherein the volume of the sample in the sample layer in the assay region is approximately equal to the product of the predetermined assay region and the predetermined spacer height.
[Invention 1274]
The first plate comprises a first assay region and a second assay region, and the method comprises assaying the same analyte in the first assay region and the second assay region. One of 1271-1273.
[Invention 1275]
The method of any of claims 1271-1274 wherein the analyte assay region is between a pair of electrodes.
[Invention 1276]
The method of any of claims 1271-1275 wherein the assay region is defined by one patch of dry reagent.
[Invention 1277]
127. The method of any of 1271-1276 of the invention, wherein the assay region binds and immobilizes the analyte.
[Invention 1278]
The invention 1271-, wherein the assay region is defined by a patch of binding reagent, which upon contact with the sample dissolves in the sample, diffuses into the sample and binds the analyte. Either way in 1277.
[Invention 1279]
The first plate further has a plurality of analyte assay sites on its surface, the distance between the edges of two adjacent assay sites being equal to the uniform thickness when the plate is in the closed position. Substantially greater than the layer thickness, at least a portion of the layer of uniform thickness is above the assay site, and the sample has one or more analytes that can diffuse into the sample, The method of any of 1271-1278 of the present invention.
[Invention 1280]
The method of invention 1279, wherein at least two of the adjacent sites are not separated from one another to prevent analyte diffusion between the sites.
[Invention 1281]
The method of any of claims 1271-1280 of the present invention wherein the thickness of the sample is less than 200 μm.
[Invention 1282]
127. The method of any of claims 1271-1281, wherein the first plate and the second plate include a plurality of storage sites in opposing positions when the plate is in the closed configuration.
[Invention 1283]
The method of any of claims 1271-1282 wherein the ratio of the linear dimension of the binding site to the uniform thickness is greater than 5.
[Invention 1284]
The method of any of claims 1271-1283 wherein the reaction is saturated in less than 60 seconds.
[Invention 1285]
The method of any of claims 1271-1284, wherein said relevant time is in the range of 60 seconds to 30 minutes.
[Invention 1286]
In step e(i), the method of any of the inventions 1271-1285, comprising assessing the target entity bound to the binding site after stopping the incubation.
[Invention 1287]
127. The method of any of 1271-1286 of the present invention, comprising one or more washes.
[Invention 1288]
The method according to any of the inventions 1271 to 1287, wherein the distance between the spacers is in the range of 14 μm to 200 μm.
[Invention 1289]
The method according to any one of the inventions 1271 to 1288, wherein the distance between the spacers is in the range of 7 μm to 20 μm.
[Invention 1290]
Any of the inventions 1271-1289, wherein the spacer is a column having a cross-sectional shape selected from a circle, a polygon, a ring, a square, a rectangle, an oval, an ellipse, or any combination thereof. Method.
[Invention 1291]
Any of the inventions 1271-1290, wherein said spacers have a pillar shape and have a substantially flat upper surface, and for each spacer the ratio of its lateral dimension to the height of said spacer is at least 1. That way.
[Invention 1292]
The method according to any one of the inventions 1271 to 1291, wherein the spacer has a pillar shape, and a sidewall angle of the spacer has a circular shape having a radius of curvature of at least 1 μm.
[Invention 1293]
The spacer has a density of at least 1000/mm ² The method of any of 1271-1292 of the present invention which is:
[Invention 1294]
The method of any of claims 1271-1293 wherein at least one of the plates is transparent.
[Invention 1295]
The method of any of claims 1271-1294 wherein at least one of the plates is made of a flexible polymer.
[Invention 1296]
The method of any of claims 1271-1295 of the present invention wherein only one of the plates is flexible.

は血液試料を使用する１７５μｍ厚のＰＭＭＡ用の標識であり、標識

は唾液試料を使用する１７５μｍ厚のＰＭＭＡ用の標識であり、標識

はＰＢＳ試料を使用する１２５μｍ厚のＰＳ用の標識であり、標識

は血液試料を使用する５０μｍ厚のＰＭＭＡ用の標識であり、標識

は血液試料を使用する２５μｍ厚のＰＳ用の標識である。
（図１９）図１９は、Ｘ‐プレートのＩＳＤ^４／（ｈ^ｘＥ）（概略図中、ｘ＝１）値に対する、試料の厚さの偏差及び均一性の測定値を示す。ＩＳＤはスペーサ間の距離であり、ｈは材料の高さ（厚さ）であり、Ｅは材料のヤング率であり、ｘは典型的な範囲が１〜３のフィッティングパラメータである。該テストでは、ＣＲＯＦ装置の基板は、２５０μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、Ｘ‐プレートは１７５μｍ厚の未処理ＰＭＭＡ、１２５μｍ厚の未処理ＰＳ、５０μｍ厚の未処理ＰＭＭＡ、及び２５μｍ厚の未処理ＰＳ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、それは、周期的な柱状スペーサアレイを含み、スペーサの高さが５μｍであり、形状が長方形であり（柱の横方向のサイズが１０×１０μｍであり、断面がほとんど均一であり、角が丸められる）、スペーサ間の距離が２０μｍ、５０μｍ、１００μｍ、２００μｍ、５００μｍであり、試料は、２μＬの血液（指で直接触れて滴下する）、唾液又はＰＢＳ（ピペットで滴下する）であり、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。ＩＳＤ^４／ｈ^ｘ＝１／Ｅの計算において、ＰＭＭＡのヤング率は２．５ＧＰａ、ＰＳのヤング率は３．３ＧＰａである。ＩＳＤ^４／（ｈＥ）の値が１０^６μｍ^３／ＧＰａより大きい場合、ＣＲＯＦ装置の性能は悪化する。図面において、標識

は血液試料を使用する１７５μｍ厚のＰＭＭＡ用の標識であり、標識

は唾液試料を使用する１７５μｍ厚のＰＭＭＡ用の標識であり、標識

はＰＢＳ試料を使用する１２５μｍ厚のＰＳ用の標識であり、標識

は血液試料を使用する５０μｍ厚のＰＭＭＡ用の標識であり、標識

は血液試料を使用する２５μｍ厚のＰＳ用の標識である。
（図２０）図２０は、Ｘ−プレート上の異なる柱状スペーサのサイズ及び高さでのスペーサ間の距離に対する、試料の厚さの偏差及び均一性の測定値を示す。ＣＲＯＦ装置の基板は、１ｍｍ厚の未処理ガラス（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、Ｘ‐プレートは１２５μｍ厚の未処理ＰＳ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、それは、スペーサの高さが５μｍであり、形状が長方形であり（柱の横方向のサイズが１０×１０μｍであり、断面がほとんど均一であり、角が丸められる）、スペーサ間の距離が２０μｍ、５０μｍ、１００μｍ、２００μｍ、５００μｍである周期的な柱状スペーサアレイ

と、柱の横方向のサイズが４０×４０μｍであり、スペーサ間の距離が６０μｍ、１５０μｍ、及び２００μｍである周期的な柱状スペーサアレイ

と、スペーサの高さが１２μｍであり、形状が長方形であり、柱の横方向のサイズが４０×４０μｍであり、スペーサ間の距離が１５０μｍ、２００μｍである周期的な柱状スペーサアレイ

と、スペーサの高さが２２μｍであり、形状が長方形であり、柱の横方向のサイズが４０×４０μｍであり、スペーサ間の距離が１５０μｍ、２００μｍである周期的な柱状スペーサアレイ

とを含み、試料は、ＰＢＳ（ピペットで滴下する）であり、５μｍ厚のＣＲＯＦの場合は２μＬの試料を使用し、１２μｍ厚のＣＲＯＦの場合は５μＬの試料を使用し、２２μｍ厚のＣＲＯＦの場合は９μＬの試料を使用し、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。（図中の線は視線誘導のためのものである。）
（図２１）図２１は、全ての１５０μｍ未満の全試料についてＩＳＤを保持する間の、柱の高さに対する柱の幅の異なる比に対する、試料の厚さの偏差及び均一性の測定値を示す。ＣＲＯＦ装置の基板は、１ｍｍ厚の未処理ガラス（サイズが２５．４ｍｍ×２５．４ｍｍである）である。ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。上図における標識付きの試料は以下のとおりである。
Ａ．（標識

の付いた）１２５μｍ厚のＰＳで製造されたＸ‐プレート、左から右へ：１：Ｘ‐プレート柱サイズ１０×１０μｍ、高さ２２μｍ、ＩＳＤ １００μｍ、９μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝０．４５；２：Ｘ‐プレート柱サイズ１０×１０μｍ、高さ１２μｍ、ＩＳＤ １００μｍ、５μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝０．８３；３：Ｘ‐プレート柱サイズ４０×４０μｍ、高さ２２μｍ、ＩＳＤ １５０μｍ、９μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝１．８１；４：Ｘ‐プレート柱サイズ４０×４０μｍ、高さ５μｍ、ＩＳＤ １００μｍ、２μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝２；５：Ｘ‐プレート柱サイズ４０×４０μｍ、高さ１２μｍ、ＩＳＤ １５０μｍ、５μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝３．３３；６：Ｘ‐プレート柱サイズ４０×４０μｍ、高さ５μｍ、ＩＳＤ １５０μｍ、２μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝８；７：Ｘ‐プレート柱サイズ７０×７０μｍ、高さ５μｍ、ＩＳＤ １５０μｍ、２μＬ ＰＢＳ緩衝液、比（ｗ／ｈ）＝１４。
Ｂ．（標識

の付いた）１７５μｍ厚のＰＭＭＡで製造されたＸ‐プレート、左から右へ：１：Ｘ‐プレート柱サイズ１０×１０μｍ、高さ２２μｍ、ＩＳＤ １００μｍ、５μＬ 血液、比（ｗ／ｈ）＝０．４５；２：Ｘ‐プレート柱サイズ１０×１０μｍ、高さ５μｍ、ＩＳＤ ５０μｍ、２μＬ 血液、比（ｗ／ｈ）＝２；３：Ｘ‐プレート柱サイズ３０×３０μｍ、高さ３０μｍ、ＩＳＤ ８０μｍ、１２μＬ 血液、比（ｗ／ｈ）＝１；４：Ｘ‐プレート柱サイズ３０×３０μｍ、高さ１０μｍ、ＩＳＤ ８０μｍ、１μＬ 血液、比（ｗ／ｈ）＝３；５：Ｘ‐プレート柱サイズ３０×３０μｍ、高さ２μｍ、ＩＳＤ ８０μｍ、１μＬ 血液、比（ｗ／ｈ）＝１５。
Ｃ．（標識

の付いた）５０μｍ厚のＰＭＭＡで製造されたＸ‐プレート、左から右へ：１：Ｘ‐プレート柱サイズ１０×１０μｍ、高さ５μｍ、ＩＳＤ ５０μｍ、２μＬ 血液、比（ｗ／ｈ）＝２。
Ｄ．（標識

の付いた）２５μｍ厚のＰＳで製造されたＸ‐プレート、左から右へ：１：１：Ｘ‐プレート柱サイズ１０×１０μｍ、高さ５μｍ、ＩＳＤ ５０μｍ、２μＬ 血液、比（ｗ／ｈ）＝２。
（図２２）図２２は、Ｘ‐プレートのスペーサ間の距離及び柱のサイズ／高さに対する、試料の厚さの偏差及び均一性の測定値を示し、ＣＲＯＦ装置の基板は、１ｍｍ厚の未処理ガラス（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、Ｘ‐プレートは１２５μｍ厚の未処理ＰＳ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、それは、スペーサの高さが５μｍであり、形状が長方形であり、柱の横方向のサイズが１０×１０μｍであり（断面がほとんど均一であり、角が丸められる）、スペーサ間の距離が２０μｍ、５０μｍ、１００μｍ、２００μｍ、５００μｍである周期的な柱状スペーサアレイ

と、柱の横方向のサイズが４０×４０μｍであり、スペーサ間の距離が６０μｍ、１５０μｍ、及び２００μｍである周期的な柱状スペーサアレイ

と、スペーサの高さが１２μｍであり、形状が長方形であり、柱の横方向のサイズが４０×４０μｍであり、スペーサ間の距離が６０μｍ、１５０μｍ、及び２００μｍである周期的な柱状スペーサアレイ

と、スペーサの高さが２２μｍであり、形状が長方形であり、柱の横方向のサイズが４０×４０μｍであり、スペーサ間の距離が１５０μｍ及び２００μｍである周期的な柱状スペーサアレイ

とを含み、試料は、ＰＢＳ（ピペットで滴下する）であり、５μｍ厚のＣＲＯＦの場合は２μＬの試料を使用し、１２μｍ厚のＣＲＯＦの場合は５μＬの試料を使用し、２２μｍ厚のＣＲＯＦの場合は９μＬの試料を使用し、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。（図中の線は視線誘導のためのものである。）
（図２３）図２３は、一定の柱のサイズ（３０×３８μｍ）、柱の高さ（２μｍ）及びスペーサ間の距離（８０×８２μｍ）を有し、未処理ＰＭＭＡで製造されたＸ−プレートの異なる厚さ（２５μｍ〜５２５μｍ）に対する、試料の厚さの偏差及び均一性の測定値を示し、ここで、基板は、１ｍｍ厚の未処理ガラス（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、試料は、指で直接触れて滴下した１μＬの血液であり、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。
（図２４）図２４は、測定されたＣＲＯＦ装置の間隔サイズの偏差／均一性（標識

の付いた親水性‐親水性及び標識

の付いた親水性‐疎水性の異なる対）を示し、血液量が０．１μＬ〜０．５μＬであり、Ｘ−プレート柱サイズ（３０×３８μｍ）、柱の高さ（２μｍ）及びスペーサ間の距離（８０×８２μｍ）が同じであり、ここで、基板は１ｍｍ厚のガラス（サイズが２５．４ｍｍ×２５．４ｍｍである）であり、Ｘ−プレートは１７５μｍ厚のＰＭＭＡで製造され、サイズが２５．４ｍｍ×２５．４ｍｍである。血液は、指で直接触れて滴下し、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。
（図２５）図２５は、標識

の付いた１ｍｍ厚の未処理ガラス又は標識

の付いた２５０μｍ厚の未処理ＰＭＭＡで製造された基板（サイズは２５．４ｍｍ×２５．４ｍｍである）に対する、試料の厚さの偏差及び均一性の測定値を示し、ここで、Ｘ‐プレートは１７５μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、それは、周期的な柱状スペーサアレイを含み、スペーサの高さが５μｍであり、形状が長方形であり（柱の横方向のサイズが１０×１０μｍであり、断面がほとんど均一であり、角が丸められる）、スペーサ間の距離が５０μｍ、１００μｍ、２００μｍ、５００μｍであり、試料は、指で直接触れて滴下する２μＬの血液であり、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。
（図２６）図２６は、異なる手によるプレス時間０秒〜６０秒でのテストに対する、試料の厚さの偏差及び均一性の測定値を示し、ここで、ＣＲＯＦ装置は２５０μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、Ｘ‐プレートは１７５μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、それは、周期的な柱状スペーサアレイを含み、スペーサの高さが２μｍであり、形状が長方形であり（柱の横方向のサイズが３０×３８μｍであり、断面がほとんど均一であり、角が丸められる）、スペーサ間の距離が８０μｍであり、試料は、指で直接触れて滴下した１μＬの血液であり、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。
（図２７）図２７は、ランダムボールスペーサ又は規則的な柱状スペーサ（Ｘ‐プレート）を使用するための平均ＩＤＳに対する、試料の厚さの偏差及び均一性の測定値を示し、ここで、ＣＲＯＦ装置は１ｍｍ厚の未処理ガラス（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、Ｘ‐プレートは１７５μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）であり、それは、周期的な柱状スペーサアレイを含み、スペーサの高さが５μｍであり、形状が長方形であり（柱の横方向のサイズが１０×１０μｍであり、断面がほとんど均一であり、角が丸められる）、スペーサ間の距離が２０μｍ、５０μｍ、及び１００μｍであり、試料は、２μＬのＰＢＳであり、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。前記ボールは、ＰＢＳ中の平均直径が４μｍ（サイズ変動５％）のソーダ石灰ミクロスフェアである。マイクロスフェアは、４×１０^５／μＬ、０．９×１０^５／μＬ及び０．２×１０^５／μＬの濃度でＰＢＳ中に分布し、それぞれ２０μｍ、５０μｍ及び１００μｍのプレス後の平均スペーサ間の距離に対応する。２２０μｍ厚の未処理ガラス（サイズは２５．４ｍｍ×２５．４ｍｍである）及び１７５μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）の二種類のカバープレートを使用する。全ての装置を手でプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。標識

はＸ‐プレート用の標識であり、標識

はスペーサとしてのビーズ、カバープレートとしての２２０μｍ厚のガラスプレート用の標識であり、標識

はスペーサとしてのビーズ、カバープレートとしての１７５μｍ厚のＰＭＭＡフィルム用の標識である。
（図２８）図２８は、異なるＸ−プレートの厚さ（２５μｍ〜３５０μｍ）と基板の厚さ（２５μｍ〜７５０μｍ）に対する、試料の厚さの偏差及び均一性の測定値を示す。Ｘ‐プレートは、一定の柱のサイズ（３０×３８μｍ）、柱の高さ（１０μｍ）及びスペーサ間の距離（８０×８２μｍ）を有し、２５μｍ、１７５μｍ、及び３５０μｍ厚の未処理ＰＭＭＡで製造され、ここで、基板は、２５μｍ、５０μｍ、１７５μｍ、２５０μｍ、及び７５０μｍ厚の未処理ＰＭＭＡ（サイズは２５．４ｍｍ×２５．４ｍｍである）で製造される。試料は、指で直接触れて滴下した４μＬの血液であり、ＣＲＯＦ装置を手でハンドプレスしかつ１インチ×１インチの領域で擦り、かつプレス後に自己保持される。図において、標識

は２５μｍ厚のＸ‐プレート用の標識であり、標識

は１７５μｍ厚のＸ‐プレート用の標識であり、標識

は３５０μｍ厚のＸ‐プレート用の標識である。
（図２９）図２９は、（ａ）Ｘ‐装置における血球の顕微鏡写真（４０×）を示し、ここで、プレート間隔（したがって、試料の厚さ）は１μｍ、２μｍ、３μｍ、及び５μｍである。間隔が１μｍであるＸ‐装置は、ＲＢＣの大部分（９９％）を溶解し、血小板は溶解しない。間隔が２μｍであるＸ‐装置は、各ＲＢＣを上手く分離し、かつＲＢＣを単一層にする。間隔が３μｍであるＸ‐装置において、いくつかの積み重ねられたＲＢＣが観察され、間隔が５μｍであるＸ‐装置において、より多くの積み重ねられたＲＢＣが観察される。計数時に、単層細胞（２μｍのＸ‐装置）が好ましい。図２９（ｂ）は、さらに（ｂ）ＣＲＯＦプレートの総側面積に対する赤血球面積（２Ｄ上面図から測定）の比を示す。プレート間隔（すなわち、サンプルの厚さ）が２μｍである場合に、それは最も大きく、その原因は、２μｍ未満の場合にいくつかのＲＢＣが溶解し、２μｍより大きい場合にＲＢＣが重なりかつ回転することにより、２Ｄ画像でのＲＢＣ面積が小さくなることである。
（図３０）図３０は、スマートフォンによるＢＣＩ（ＣＲＯＦと撮像による血球計数）の概略図（ａ）及び装置の写真（ｂ）を示す。スマートフォン‐ＢＣＩを使用した血液検査では、まず１人がカードを持っていて（１）、指を刺した後（２）、カードに触れて少量の血液を指からＣＲＯＦカードに直接滴下し（２）、カードを閉じ（３）、指でプレスし（４）、それを解放して、カードを光学アダプタに挿入し（５）、最後にスマートフォンを使用して該カードを撮影して、撮影された写真から、ソフトウェアは血液量、血球数及び他のパラメータを測定する（６）。（ｂ）はリアルなスマートフォンの写真とｐ−ＢＣＩアダプタである。
（図３１）図３１は、異なる最終ギャップを有するＣＲＯＦカード中の新鮮な未希釈全血（ａ）と貯蔵された未希釈全血（ｂ）の明視野光学顕微鏡画像、及び異なる閉じ込めギャップに対応するＲＢＣ挙動の図である。刺された指から取り出された新鮮な血液は、抗凝血剤を有し、商業的供給者から貯蔵された血液は、抗凝血剤を有する。（ａ−１〜ａ−６）と（ｂ−１〜ｂ−６）において、それぞれｇ＝２、２．２、２．６、３、５及び１０μｍである。（ｃ）は、横断面と平面図の概略図を示し、（１）ＲＢＣが相互に分離し、２μｍのギャップのＣＲＯＦにおいて観察可能な重なりを有しないことと、（２）ギャップが２μｍよりも大きいＣＲＯＦにおいてＲＢＣが互いに重なることとを示す。
（図３２）図３２は、光学アダプタを備えたスマートフォン（ａ）とデジタル一眼（ＤＳＬＲ）カメラを備えた高解像度顕微鏡（ｂ）によって撮影された同じ試料（ＣＲＯＦカード中の撮影した新鮮な血液）の明視野（１）と蛍光（２）画像である。画像から分かるように、光学アダプタを備えたスマートフォンは、高解像度顕微鏡及びカメラに類似する血球写真の品質を有する。
（図３３）図３３は、典型的なＷＢＣ及びＰＬＴの計測された光強度とそれらの分離細胞の位置との比較を示す。ＷＢＣの直径（ＦＷＨＭ）は約１２μｍであり、ＰＬＴの直径（ＦＷＨＭ）は約２μｍである。ＷＢＣの最大強度は、ＰＬＴより約３倍大きい。強度及び面積の両方により、ＷＢＣの全体の強度はＰＬＴより約１０８倍大きい。したがって、低い拡大倍率（例えば４×）を用いると、ＷＢＣの面積が小さくなり、全体の強度も低下する。このような場合には、ＰＬＴの信号は無視することができる。
（図３４）図３４は、（ａ）緑色チャネル光の強度と赤色チャネル強度の点分布図と、（ｂ）画像内の５９４個のＷＢＣの赤色／緑色チャネルの強度比のヒストグラムとを示す。この画像から明らかなように、細胞は３つの異なる区域（指向として陰影区域に示される）に集合し、３つの主な白血球サブセットに対応する。 Those skilled in the art will appreciate that the drawings, described below, are for illustration purposes only. The drawings are not intended to limit the scope of the present teachings in any way. Drawings may not be drawn to scale. In the drawings showing the current experimental data points, the lines connecting the data points are only for guiding the browsing of the data, not other means.
FIG. 1 is a diagram of an embodiment of CROF (Compression Controlled Open Flow). FIG. 1A shows a first plate having a spacer and a second plate. Figure (b) shows depositing the sample on the first plate (shown) or the second plate (not shown) or both (not shown) in the open configuration. Figure (c) shows (i) spreading the sample with two plates (sample flowing between the plates) to reduce the thickness of the sample, and (ii) a closed configuration with spacers and plates. And adjusting the thickness of the sample in FIG. The inner surface of each plate may have one or more binding sites and/or storage sites (not shown).
FIG. 2 shows a plate with binding or storage sites. Figure (a) shows a plate with binding sites. Figure (b) shows a plate with reagent storage sites. Figure (c) shows a first plate with binding sites and a second plate with reagent storage sites. FIG. (d) shows a plate having a plurality of sites (binding site and/or storage site).
(FIG. 3) FIG. 3 is a flow chart and schematic of a method for reducing assay incubation time by reducing sample thickness. Figure (a) shows a first plate having at least one binding site on the substrate surface. Figure (b) shows a second plate (which may have different dimensions than the first plate). Figure (c) shows depositing the sample (including target binding entity) onto the substrate surface (shown) or cover plate (not shown) or both (not shown). Figure (d) shows that the thickness of the sample is reduced by moving the first and second plates towards each other and reducing the spacing between the plates. Incubate the sample with reduced thickness. As the sample thickness decreases, the incubation time decreases. Some embodiments of the method use spacers to adjust the spacing and are not shown.
(FIG. 4) FIG. 4 shows the use of two plates, spacers and compression (shown in cross-section) to reduce the thickness of the sample to reduce binding or mixing time. Figure (a) shows shortening the time for binding entities in the sample (X- (volume to surface)) to the binding sites on the solid surface. Figure (b) shows reducing the time for binding entities (eg, reagents) stored on the surface of the plate to binding sites on another surface (X- (from surface to another surface)). .. Figure (c) shows reducing the time to add (X-(surface to volume)) the reagent stored on the surface of the plate to the sample sandwiched between the plate and the other plate. ..
(FIG. 5) FIG. 5 illustrates a method of avoiding or reducing local curvature in a flexible plate. Figure (a) shows that, under a given sample and compression conditions, the distance between the spacers is too large and the first plate is rigid with respect to the flexible plate (second plate, eg plastic film). Suppose that the plate has a local slack (ie, curved inward) between two adjacent spacers in the closed configuration. Samples between plates are not drawn. Figure (b) shows that by using suitable spacer distances and suitable compressive forces, the local curvature (slack) of the flexible plate of Figure (a) is reduced or substantially avoided. Show. Samples between plates are not drawn.
(FIG. 6) FIG. 6 illustrates reducing the effect of large dust particles on the adjustment of plate spacing (sample thickness). Figure (a) shows that when using two rigid plates, dust with a thickness greater than the height of the spacers can destroy the intended plate spacing adjustment by the spacers (and thus the intended sample. The thickness adjustment is destroyed). Samples between plates are not drawn. FIG. (b) uses a suitable flexible plate and a suitable distance between spacers to isolate the effect of dust to a small area around the dust, with plate spacing (and thus The sample thickness) indicates that it is adjusted by spacers, not dust. The first plate in this figure is rigid, the second plate is flexible and the spacers are initially fixed onto the first plate. Figure (c) uses a suitable distance between the flexible plate and a suitable spacer to isolate the effect of dust to a small area around the dust, with plate spacing (and thus The sample thickness) indicates that it is adjusted by spacers, not dust. The first plate in this figure is rigid, the second plate is flexible and the spacers are initially fixed onto the second plate.
(FIG. 7) FIG. 7 illustrates the use of suitable spacer placement and flexible plate(s) to reduce the effects of plate surface flatness variations. Figure (a) shows that the surface flatness variation is very large compared to the desired sample thickness, which causes an error in determining the sample thickness. In this figure, only one plate has large flatness variations (in fact, both plates have large flatness variations). Samples between plates are not drawn. Figure (b) shows the surface flatness variation distance of the plate, λ, which is the distance from a local maximum of surface height to an adjacent local minimum. Figure (c) shows how to make one or both of the two plates flexible and first when they are in the open configuration in the closed configuration using the appropriate spacer distance and the appropriate pressure. It is shown whether the small surface flatness variation can be realized by correcting the surface flatness variation. Samples between plates are not drawn. Figure (d) shows that by using a distance between the flexible second plate and a suitable spacer, the thickness variation of the sample is made smaller than the initial surface flatness variation of the plate. The contours of the flexible and rigid plates match. Samples between plates are not drawn.
(FIG. 8) FIG. 8 shows a plate and a closed spacer (well) for adjusting the thickness of the sample. Figure (a) shows a first plate and a second plate, where the first plate has hermetic spacers (wells). Figure (b) shows depositing the sample on the first plate (shown) or the second plate (not shown) or both (not shown) in the open configuration. Figure (c) shows (i) using two plates to spread the sample (sample flows between the plates) and reduce the thickness of the sample, and (ii) a closed configuration using spacers and plates. It is shown that the thickness of the sample is adjusted.
(FIG. 9) FIG. 9 shows another embodiment in which a sealed spacer (well) is used to adjust the thickness of a sample. Figure (a) shows a first plate and a second plate, wherein the first plate has a hermetic spacer (well) and has at least one spacer in the well. Figure (b) shows depositing the sample on the first plate (shown) or the second plate (not shown) or both (not shown) in the open configuration. Figure (c) shows (i) using two plates to spread the sample (sample flows between the plates) and reduce the thickness of the sample, and (ii) a closed configuration using spacers and plates. It shows adjusting the thickness of the sample in. Figure (d) shows another embodiment of the first plate and the second plate, where the wells of the first plate are free of spacers.
(FIG. 10) FIG. 10 schematically illustrates an exemplary embodiment of the present invention, ie, multiplex detection in a single CROF device using one binding site on one plate and multiple storage sites on another plate. Show. Figures (a) and (b) are perspective and cross-sectional views, respectively, of an exemplary device.
(FIG. 11) FIG. 11 schematically illustrates a further exemplary embodiment of the present invention, namely, multiplex detection in a single CROF device using one storage site on one plate and multiple binding sites on another plate. To indicate. Figures (a) and (b) are perspective and cross-sectional views, respectively, of an exemplary device.
(FIG. 12) FIG. 12 is a further exemplary embodiment of the present invention, namely, multiplex detection in a single CROF device using multiple binding sites on one plate and multiple corresponding storage sites on another plate. Is schematically shown. Figures (a) and (b) are perspective and cross-sectional views, respectively, of an exemplary device.
(FIG. 13A) FIG. 13A schematically illustrates a QMAX assay using CROF with a spacer array of 30 μm spacer height to achieve an assay with a saturation incubation time of less than 30 seconds.
(FIG. 13B) FIG. 13B is a measurement of the signal of captured label versus incubation time, showing that the saturation incubation time of the QMAX assay described in FIG. 13A is less than 30 seconds.
(FIG. 14) FIG. 14 shows experimentally measured LoD() for QAX and QMAX assays with a 30 μm gap (against the CROF instrument) with and without wash (heterogeneous assay). Detection limit).
(FIG. 15) FIG. 15 shows a top view and a sectional view when (i) a small amount of sample is dropped on a glass substrate and (ii) the sample region is expanded in a closed configuration of CROF.
(FIG. 16) FIG. 16 illustrates the meaning of some terms used herein.
(FIG. 17) FIG. 17 is a spacer in a plate. (A) A columnar spacer size is 46 μm×46 μm and the distance between columns is 54 μm, and (b) A columnar spacer size is 10 μm×70 μm and the distance between columns is 10 μm. Yes, (c) The size of the columnar spacer is 30 μm×40 μm and the height of the spacer is 2 μm, and (d) The size of the columnar spacer is 30 μm×40 μm and the height of the spacer is 30 μm. It is SEM.
FIG. 18 shows the effect of IDS, plate thickness and material on sample thickness. FIG. 7 shows measurements of sample thickness deviation and uniformity for different plate and spacer materials, different plate thickness and distance between spacers (IDS) for different samples. The substrate of the CROF device is a 250 μm thick untreated planar PMMA (size is 25.4 mm×25.4 mm). The X-plate includes a periodic columnar spacer array, the spacer height is 5 μm, the shape is rectangular (the column lateral size is 10×10 μm, the cross section is almost uniform, Are rounded), the distances between the spacers are 20 μm, 50 μm, 100 μm, 200 μm, 500 μm, and are made of PMMA or PS with a size of 25.4 mm×25.4 mm. The sample is 2 μL of blood (dropped by direct finger touch), saliva or PBS (dropped by pipette), hand-pressing the CROF device by hand and rubbing in a 1 inch x 1 inch area, and after pressing Self-held. In the figure, signs

Is a label for 175 μm thick PMMA using a blood sample,

Is a label for 175 μm thick PMMA using saliva samples,

Is a label for 125 μm thick PS using PBS sample,

Is a label for 50 μm thick PMMA using a blood sample,

Is a label for 25 μm thick PS using a blood sample.
(FIG. 19) FIG. 19 shows ISD of X-plate. ^Four /(H ^x E) Measured values of deviation and uniformity of the thickness of the sample with respect to the value (x=1 in the schematic). ISD is the distance between the spacers, h is the height (thickness) of the material, E is the Young's modulus of the material, and x is a fitting parameter in the typical range of 1-3. In the test, the substrate of the CROF device is 250 μm thick untreated PMMA (size is 25.4 mm×25.4 mm), the X-plate is 175 μm thick untreated PMMA, 125 μm thick untreated PS, 50 μm thick untreated PMMA and 25 μm thick untreated PS (size is 25.4 mm×25.4 mm), which comprises a periodic columnar spacer array, the spacer height is 5 μm, The shape is rectangular (the size of the column in the lateral direction is 10×10 μm, the cross section is almost uniform, and the corners are rounded), the distance between the spacers is 20 μm, 50 μm, 100 μm, 200 μm, 500 μm, and the sample Is 2 μL of blood (dropped by direct finger touch), saliva or PBS (dropped with pipette), hand-press the CROF device by hand and rub in a 1 inch×1 inch area, and self-press after pressing. Retained. ISD ^Four /H ^x=1 In the calculation of /E, PMMA has a Young's modulus of 2.5 GPa and PS has a Young's modulus of 3.3 GPa. ISD ^Four The value of /(hE) is 10 ⁶ μm ^Three If it is larger than /GPa, the performance of the CROF device deteriorates. Sign in the drawing

Is a label for 175 μm thick PMMA using a blood sample,

Is a label for PMMA of 175 μm thickness using saliva sample,

Is a label for 125 μm thick PS using a PBS sample,

Is a label for 50 μm thick PMMA using a blood sample,

Is a label for 25 μm thick PS using a blood sample.
(FIG. 20) FIG. 20 shows measurements of sample thickness deviation and uniformity versus distance between spacers at different columnar spacer sizes and heights on the X-plate. The substrate of the CROF device is 1 mm thick untreated glass (size is 25.4 mm x 25.4 mm) and the X-plate is 125 μm thick untreated PS (size is 25.4 mm x 25.4 mm). ), which has a spacer height of 5 μm, a rectangular shape (the lateral size of the pillar is 10×10 μm, the cross section is almost uniform, and the corners are rounded), and between the spacers. Periodic columnar spacer array with distances of 20 μm, 50 μm, 100 μm, 200 μm, 500 μm

And a columnar spacer array in which the lateral size of the columns is 40×40 μm and the distances between the spacers are 60 μm, 150 μm, and 200 μm.

And the spacer height is 12 μm, the shape is rectangular, the lateral size of the columns is 40×40 μm, and the distance between the spacers is 150 μm, 200 μm.

And the spacer height is 22 μm, the shape is rectangular, the lateral size of the columns is 40×40 μm, and the distance between the spacers is 150 μm, 200 μm.

The sample is PBS (dropped with a pipette), 2 μL of sample is used for 5 μm thick CROF, 5 μL of sample is used for 12 μm thick CROF, and 22 μm of CROF is used. In this case, using 9 μL of sample, the CROF device is hand-pressed by hand and rubbed in an area of 1 inch×1 inch and self-held after pressing. (The lines in the figure are for guiding the eyes.)
(FIG. 21) FIG. 21 shows measurements of sample thickness deviation and uniformity for different ratios of column width to column height while retaining ISD for all samples less than 150 μm. . The substrate of the CROF device is 1 mm thick untreated glass (size is 25.4 mm x 25.4 mm). The CROF device is hand-pressed by hand and rubbed in a 1 inch by 1 inch area and self-held after pressing. The labeled samples in the above figure are as follows.
A. (Sign

125 μm thick PS-made X-plate, left to right: 1:X-plate column size 10×10 μm, height 22 μm, ISD 100 μm, 9 μL PBS buffer, ratio (w/h). = 0.45; 2: X-plate column size 10 x 10 μm, height 12 μm, ISD 100 μm, 5 μL PBS buffer, ratio (w/h)=0.83; 3: X-plate column size 40×40 μm, Height 22 μm, ISD 150 μm, 9 μL PBS buffer, ratio (w/h)=1.81; 4: X-plate column size 40×40 μm, height 5 μm, ISD 100 μm, 2 μL PBS buffer, ratio (w/h h)=2; 5: X-plate column size 40×40 μm, height 12 μm, ISD 150 μm, 5 μL PBS buffer, ratio (w/h)=3.33; 6: X-plate column size 40×40 μm, Height 5 μm, ISD 150 μm, 2 μL PBS buffer, ratio (w/h)=8; 7: X-plate column size 70×70 μm, height 5 μm, ISD 150 μm, 2 μL PBS buffer, ratio (w/h) = 14.
B. (Sign

X-plate made of 175 μm thick PMMA (marked), left to right: 1:X-plate column size 10×10 μm, height 22 μm, ISD 100 μm, 5 μL blood, ratio (w/h)=0 .45; 2: X-plate column size 10×10 μm, height 5 μm, ISD 50 μm, 2 μL blood, ratio (w/h)=2; 3: X-plate column size 30×30 μm, height 30 μm, ISD 80 μm , 12 μL blood, ratio (w/h)=1; 4: X-plate column size 30×30 μm, height 10 μm, ISD 80 μm, 1 μL blood, ratio (w/h)=3; 5: X-plate column size 30×30 μm, height 2 μm, ISD 80 μm, 1 μL blood, ratio (w/h)=15.
C. (Sign

X-plate made of 50 μm thick PMMA (marked), left to right: 1:X-plate column size 10×10 μm, height 5 μm, ISD 50 μm, 2 μL blood, ratio (w/h)=2 ..
D. (Sign

X-plate manufactured with 25 μm thick PS (marked), left to right: 1:1:X-plate column size 10×10 μm, height 5 μm, ISD 50 μm, 2 μL blood, ratio (w/h) =2.
(FIG. 22) FIG. 22 shows measured values of deviation and uniformity of sample thickness with respect to distance between spacers of X-plate and size/height of pillars. Treated glass (size is 25.4 mm x 25.4 mm), X-plate is 125 μm thick untreated PS (size is 25.4 mm x 25.4 mm), which is the height of the spacer. Is 5 μm, the shape is rectangular, the lateral size of the column is 10×10 μm (the cross section is almost uniform, and the corners are rounded), and the distance between the spacers is 20 μm, 50 μm, 100 μm, 200 μm, 500 μm periodic columnar spacer array

And a columnar spacer array in which the lateral size of the columns is 40×40 μm and the distances between the spacers are 60 μm, 150 μm, and 200 μm.

And the spacer height is 12 μm, the shape is rectangular, the lateral size of the columns is 40×40 μm, and the distance between the spacers is 60 μm, 150 μm, and 200 μm.

And the spacer height is 22 μm, the shape is rectangular, the lateral size of the columns is 40×40 μm, and the distance between the spacers is 150 μm and 200 μm.

The sample is PBS (dropped with a pipette), 2 μL of sample is used for 5 μm thick CROF, 5 μL of sample is used for 12 μm thick CROF, and 22 μm of CROF is used. In this case, using 9 μL of sample, the CROF device is hand-pressed by hand and rubbed in an area of 1 inch×1 inch and self-held after pressing. (The lines in the figure are for guiding the eyes.)
(FIG. 23) FIG. 23 shows an X-plate made of untreated PMMA with constant post size (30×38 μm), post height (2 μm) and spacer spacing (80×82 μm). Shows thickness deviations and uniformity measurements of the sample for different thicknesses (25 μm to 525 μm), where the substrate is 1 mm thick untreated glass (size is 25.4 mm×25.4 mm). ) And the sample is 1 μL of blood dropped by direct finger touch, hand-pressing the CROF device by hand and rubbing in a 1 inch×1 inch area, and self-held after pressing.
(FIG. 24) FIG. 24 shows deviation/uniformity of the measured CROF device spacing size (marking).

Hydrophilic-hydrophilic and labeled

Hydrophilicity-hydrophobicity different pairs) with blood volume of 0.1 μL to 0.5 μL, X-plate column size (30×38 μm), column height (2 μm) and between spacers The distances (80×82 μm) are the same, where the substrate is 1 mm thick glass (size is 25.4 mm×25.4 mm) and the X-plate is made of 175 μm thick PMMA and the size is It is 25.4 mm x 25.4 mm. Blood is dropped by direct finger contact, hand-pressed on the CROF device and rubbed in a 1 inch x 1 inch area, and self-supported after pressing.
(FIG. 25) FIG. 25 shows signs

1mm thick untreated glass or sign with

FIG. 7 shows measurements of sample thickness deviation and uniformity for a substrate made of untreated PMMA with a thickness of 250 μm (size is 25.4 mm×25.4 mm), where X-plate Is a 175 μm thick unprocessed PMMA (size is 25.4 mm×25.4 mm), which contains a periodic columnar spacer array, the spacer height is 5 μm, and the shape is rectangular (columnar). Has a size in the lateral direction of 10×10 μm, its cross section is almost uniform, and its corners are rounded), the distance between spacers is 50 μm, 100 μm, 200 μm, 500 μm, and the sample is dropped by directly touching it with a finger 2 μL of blood, hand-pressing the CROF device by hand and rubbing in an area of 1 inch×1 inch, and self-holding after pressing.
(FIG. 26) FIG. 26 shows measurements of sample thickness deviations and uniformity for tests with different hand pressing times of 0 seconds to 60 seconds, where the CROF device is 250 μm thick of untreated PMMA. (Size is 25.4 mm x 25.4 mm) and the X-plate is 175 μm thick unprocessed PMMA (size is 25.4 mm x 25.4 mm), which is a periodic columnar spacer array. The height of the spacer is 2 μm, the shape is rectangular (the size of the column in the lateral direction is 30×38 μm, the cross section is almost uniform, and the corners are rounded), and the distance between the spacers is 80 μm. And the sample is 1 μL of blood dropped by direct finger contact, hand-pressing the CROF device by hand and rubbing in a 1 inch×1 inch area, and self-holding after pressing.
FIG. 27 shows measurement of sample thickness deviation and uniformity versus average IDS for using random ball spacers or regular columnar spacers (X-plates), where CROF The device is 1 mm thick untreated glass (size is 25.4 mm x 25.4 mm), the X-plate is 175 μm thick untreated PMMA (size is 25.4 mm x 25.4 mm), It contains a periodic array of columnar spacers, the spacer height is 5 μm, the shape is rectangular (the column lateral size is 10×10 μm, the cross section is almost uniform, and the corners are rounded). ), the distances between the spacers are 20 μm, 50 μm, and 100 μm, the sample is 2 μL PBS, hand-pressed the CROF device by hand and rubbed in a 1 inch×1 inch area, and self-held after pressing. It The balls are soda-lime microspheres with an average diameter of 4 μm in PBS (size variation 5%). Microsphere is 4×10 ⁵ /ΜL, 0.9×10 ⁵ /ΜL and 0.2×10 ⁵ Distributed in PBS at a concentration of /μL, corresponding to the distance between the average spacers after pressing of 20 μm, 50 μm and 100 μm, respectively. Two types of cover plates are used: 220 μm thick untreated glass (size is 25.4 mm×25.4 mm) and 175 μm thick untreated PMMA (size is 25.4 mm×25.4 mm). All equipment is pressed by hand and rubbed in a 1 inch x 1 inch area and self-held after pressing. Sign

Is the label for the X-plate,

Is a bead as a spacer, a label for a 220 μm thick glass plate as a cover plate,

Is a bead as a spacer and a label for a 175 μm thick PMMA film as a cover plate.
(FIG. 28) FIG. 28 shows measured deviations and uniformity of sample thickness for different X-plate thickness (25 μm to 350 μm) and substrate thickness (25 μm to 750 μm). X-Plates have constant post size (30x38μm), post height (10μm) and spacer spacing (80x82μm) and are made of 25μm, 175μm and 350μm thick unprocessed PMMA Where the substrate is made of 25 μm, 50 μm, 175 μm, 250 μm, and 750 μm thick untreated PMMA (size is 25.4 mm×25.4 mm). The sample is 4 μL of blood dropped by direct finger touch, hand-pressing the CROF device by hand and rubbing in a 1 inch×1 inch area, and self-held after pressing. In the figure, signs

Is a label for 25 μm thick X-plate,

Is a label for a 175 μm thick X-plate,

Is a label for a 350 μm thick X-plate.
(FIG. 29) FIG. 29 shows (a) a micrograph (40×) of blood cells in the X-apparatus, where the plate spacing (and therefore sample thickness) is 1 μm, 2 μm, 3 μm, and 5 μm. .. The X-device with 1 μm spacing lyses most of the RBCs (99%) and not platelets. An X-device with a spacing of 2 μm successfully separates each RBC and makes the RBCs a single layer. Some stacked RBCs are observed in the X-device with a spacing of 3 μm, and more stacked RBCs are observed in the X-device with a spacing of 5 μm. When counting, monolayer cells (2 μm X-device) are preferred. FIG. 29( b) further shows (b) the ratio of the red blood cell area (measured from a 2D top view) to the total lateral area of the CROF plate. It is greatest when the plate spacing (ie sample thickness) is 2 μm, because some RBCs dissolve below 2 μm and RBCs overlap and rotate above 2 μm. This reduces the RBC area in the 2D image.
(FIG. 30) FIG. 30 shows a schematic diagram (a) of a BCI (CROF and blood cell count by imaging) by a smartphone and a photograph (b) of the apparatus. In a blood test using a smartphone-BCI, one person first had a card (1), stabbed his finger (2), then touched the card and dripped a small amount of blood directly from his finger onto the CROF card (2). ), close the card (3), press with your finger (4), release it, insert the card into the optical adapter (5), and finally take a picture of the card with your smartphone From the photographs taken, the software measures blood volume, blood cell counts and other parameters (6). (B) is a photograph of a realistic smartphone and a p-BCI adapter.
(FIG. 31) FIG. 31 corresponds to bright field optical microscopy images of fresh undiluted whole blood (a) and stored undiluted whole blood (b) in CROF cards with different final gaps, and different confinement gaps. FIG. 5 is a diagram of RBC behavior of the subject. Fresh blood drawn from a pricked finger has anticoagulant and blood stored from a commercial supplier has anticoagulant. In (a-1 to a-6) and (b-1 to b-6), g=2, 2.2, 2.6, 3, 5, and 10 μm, respectively. (C) shows a schematic view of a cross-section and a plan view, (1) the RBCs are separated from each other and have no observable overlap in the CROF of the 2 μm gap, and (2) the gap is larger than 2 μm. It is shown that RBCs overlap each other in a large CROF.
(FIG. 32) FIG. 32 shows the same sample (fresh blood taken in a CROF card) taken by a smartphone with optical adapter (a) and a high-resolution microscope (b) with a digital single-lens (DSLR) camera. And bright field (1) and fluorescence (2) images. As can be seen from the images, the smartphone with the optical adapter has a hemocytograph quality similar to a high resolution microscope and camera.
(FIG. 33) FIG. 33 shows a comparison of the measured light intensities of typical WBCs and PLTs with their isolated cell locations. The WBC diameter (FWHM) is about 12 μm and the PLT diameter (FWHM) is about 2 μm. The maximum intensity of WBC is about 3 times higher than PLT. Due to both strength and area, the overall strength of WBC is about 108 times greater than PLT. Therefore, using a low magnification (eg 4×) will reduce the WBC area and reduce the overall strength. In such a case, the PLT signal can be ignored.
(FIG. 34) FIG. 34 shows (a) a point distribution diagram of the green channel light intensity and the red channel intensity, and (b) a histogram of the intensity ratio of the red/green channels of 594 WBCs in the image. As is evident from this image, cells aggregate in three different areas (shown as shaded areas for orientation), corresponding to the three major leukocyte subsets.

Detailed Description of Illustrative Embodiments The following detailed description illustrates some embodiments of the invention by way of non-limiting example. Section headings and any subtitles used herein are for organizational purposes only and are not to be understood as limiting the described subject matter in any way. The content of section headings and/or subtitles is not limited to section headings and/or subtitles, but applies to the overall description of the invention.

  Citation of any publication is for purposes of disclosure prior to the filing date of the present application and is not to be construed as an admission that the invention is not entitled to antedate such publication by virtue of prior invention. Also, the date of publication provided may differ from the actual date of publication, which must be independently confirmed.

  The present invention relates in particular to methods, devices and systems capable of improving and/or speeding up the quantification, binding and/or detection of analytes and/or entities in a sample.

Definitions Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this disclosure belongs. Although some exemplary methods and materials are described below, any similar or identical methods and materials described herein may be used in the practice or testing of the present teachings.

  The terms “polypeptide”, “peptide”, and “protein” refer to polymers of amino acids of any length and may be used interchangeably herein. The polymer may be linear or branched, it may comprise modified amino acids, and it may be blocked by non-amino acids. The term further includes amino acid polymers modified by other manipulations such as, for example, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation or conjugation with labeling moieties and the like. As used herein, the term "amino acid" refers to natural and/or unnatural or synthetic amino acids and includes both glycine and the D or L enantiomers, amino acid analogs and peptidomimetics.

  The terms "polynucleotide", "nucleotide", "nucleotide sequence", "nucleic acid", "nucleic acid molecule", "nucleic acid sequence", and "oligonucleotide" are used interchangeably and are each in the context of the term in which they are used. Correspondingly, plural of each term may be included. They refer to a polymeric form of nucleotides of any length and may be either deoxyribonucleotides (DNA) or ribonucleotides (RNA), or analogs thereof. The polynucleotide may have any three-dimensional structure and may perform any known or unknown function. The following are non-limiting examples of polynucleotides. Coding or non-coding region of gene or gene fragment, locus defined by linkage analysis, exon, intron, messenger RNA (mRNA), transfer RNA (tRNA), ribosomal RNA, ribozyme, small interfering RNA (siRNA), micro RNA (MiRNA), micronucleus RNA (snRNA), cDNA, recombinant polynucleotide, branched-chain polynucleotide, plasmid, carrier, isolated DNA (A, B and Z structure) having an arbitrary sequence, PNA, locked nucleic acid (LNA). ), TNA (threose nucleic acid), isolated RNA having an arbitrary sequence, a nucleic acid probe and a primer. LNA is a modified RNA nucleotide commonly referred to as inaccessible RNA. The ribose portion of the LNA nucleotide is modified by an extra bridge connecting the 2'and 4'carbons. The cross-link "locks" ribose into the 3'-terminal structure, which is commonly expressed in DNA or RNA Form A, and can significantly improve thermostability.

The term "capture agent" as used herein refers to a binding member such as, for example, a nucleic acid molecule, a polypeptide molecule or any other molecule or compound capable of specifically binding to its binding partner, eg A second nucleic acid molecule having a nucleotide sequence complementary to one nucleic acid molecule, an antibody specifically recognizing an antigen, an antigen specifically recognized by the antibody, a nucleic acid aptamer capable of specifically binding to a target molecule, etc. . The capture agent can concentrate the target molecule from a heterogeneous mixture of different molecules by specifically binding to the target molecule. The binding may be non-covalent or covalent. When specifically binding to each other in a binding complex, the affinity between the binding member and its specifically binding binding partner is such that its K ^D (dissociation constant) is 10 ⁻⁵ M or less, 10 ⁻⁶ M. ^Or less, for example 10 ⁻⁷ M or less, including 10 ⁻⁸ M or less, for example 10 ⁻⁹ M or less, 10 ⁻¹⁰ M or less, 10 ⁻¹¹ M or less, 10 ⁻¹² M or less, 10 ⁻¹³ M Hereinafter, it is 10 ^-14 M or less, 10 ^-15 M or less, and 10 ^-16 M or less is included. "Affinity" refers to the strength of binding, and increased binding affinity is associated with decreased K ^D.

  The term "secondary capture agent", also called "detection agent", refers to a group of biomolecules or chemical compounds that have a very specific affinity for an antigen. The secondary capture agent can be tightly bound to an optically detectable label (eg, an enzyme, a fluorescent label), or can itself be bound to the optically detectable label by bioconjugation to other detection agents. (Hermanson, "Bioconjugate Technologies" Academic Press, Second Edition, 2008).

  The term "scavenger-reactive group" refers to a moiety of a chemical function in a molecule that is capable of reacting with a scavenger, ie, reacting with a part in a scavenger (eg, hydroxyl, sulfhydryl, carboxyl or amine). It is one that produces stable and strong covalent bonds.

  The terms "specific binding" and "selective binding" refer to the ability of a capture agent to preferentially bind to a particular target analyte present in a heterogeneous mixture with different target analytes. Specific or selective binding interacts to discriminate between desired (eg, activated) and undesired (eg, inactivated) target analytes in a sample, generally about 10 to 100-fold (eg, More than about 1000 or 10,000 times) or more.

  The term "antibody" as used herein refers to a protein consisting of one or more polypeptides substantially encoded by all or part of a recognized immunoglobulin gene. For example, the immunoglobulin genes recognized in humans include the kappa (κ), lambda (λ) and heavy chain loci that make up a myriad of variable region genes, and the constant region genes mu (μ), delta (δ). ), gamma (γ), sigma (σ), and also includes alpha (α) encoding IgM, IgD, IgG, IgE and IgA antibodies “isotype” or “class”, respectively. Antibodies herein are meant to include full-length antibodies and antibody fragments and are naturally occurring antibodies, engineered antibodies from any organism or recombinantly produced for experimental, therapeutic or other purposes. Refers to an antibody. The term "antibody" includes full-length antibodies and antibody fragments known in the art, eg, Fab, Fab', F(ab')2, Fv, scFv, or other antigen binding subsequences of an antibody, which are It is produced by modifying a complete antibody or is newly synthesized by using recombinant DNA technology.

  The terms "antibody epitope", "epitope", "antigen" are used interchangeably herein and refer to a biomolecule bound by an antibody. Antibody epitopes may include proteins, carbohydrates, nucleic acids, hormones, receptors, tumor markers, etc. and mixtures thereof. Antibody epitope further refers to a group of antibody epitopes, such as a particular portion of a protein eluted from a size exclusion chromatography column. Also, antibody epitopes can be recognized as designated clones from expression libraries or random epitope libraries.

  As used herein, the term "allergen" is a substance in an individual that causes an allergic inflammatory response when the individual is exposed to the substance, for example by skin contact, ingestion, inhalation, eye contact, etc. Allergens include a group of substances that together cause an allergic reaction. Allergens include natural and artificial fibers (cotton, linen, wool, silk, teak, wood, straw and other dust) and tree pollen (han trees, birch trees, hazel trees, oak trees, poplar trees, Palm trees, etc., weeds and flowers (Ambrussia, Artemisia, etc.), and gramineous plants and cereal plants (Euphoria japonicus, timothy grass, rye, wheat, corn, staghorn etc.), and drugs (antibiotics, antibacterial agents, analgesics) And nonsteroidal anti-inflammatory drugs, anesthetics and muscle relaxants, hormones, etc., epidermis and animal allergens (epithelial tissue of animals, bird feathers, serum, etc.), molds and yeasts (Penicillium notation), Cladosporium spp., Aspergillus fumigatus, Mucor racemosus, etc., insect venom, preservatives (butylparaben, sorbic acid, benzoic acid, etc.), and semen (Ejaculation), and parasites and mite allergens (roundworms, Dermatophagoides pteronyssinus), Dermatophagoides farinae, crocodile mites (Euroglyphus mayne, etc.), profession, and other forms of agar (Euroglyphus maynei); ) And food allergens (egg products, dairy products and cheese, meat products, fish and seafood products, soy products, mushrooms, flour and cereals, vegetables, melons and yugao, beans, herbs and spices, nuts, citrus fruits and others. , Fruits, berries, tea and herbs, dietary supplements, etc.) and sources classified into groups.

  The term "hybridization" refers to the reaction of one or more polynucleotides to form a complex that is stabilized by hydrogen bonds between the bases of nucleotide residues. Hydrogen bonds can be generated by Watson-Crick base pairing, Hoogstein bonds or any other sequence specific manner. The complex may comprise two strands forming a double-stranded structure, three or more strands forming a multi-chain complex, a single self-hybridizing strand or any combination thereof.

  Hybridization can be performed under a variety of stringent conditions, as is known to those skilled in the art. With proper hybridization conditions, the recognition interaction between the capture sequence and the target nucleic acid is not only sufficiently specific but also sufficiently stable. Conditions that increase the stringency of hybridization reactions are well known and published in the art. See, eg, Green et al. (2012), below.

  The term “protein” refers to a polymeric form of amino acids of any length, ie, greater than 2 amino acids, greater than about 5 amino acids, greater than about 10 amino acids, greater than about 20 amino acids. Greater than about 50 amino acids, greater than about 100 amino acids, greater than about 200 amino acids, greater than about 500 amino acids, greater than about 1000 amino acids, greater than about 2000 amino acids, Generally not greater than about 10,000 amino acids, which may include coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, and polypeptides having a modified peptide backbone. The term includes fusion proteins with heterologous amino acid sequences, fusion proteins with heterologous and cognate leader sequences, with or without an N-terminal methionine residue, immunologically labeled proteins, detectable It may include a fusion protein having a fusion partner, for example, a fusion protein including, but not limited to, a fluorescent protein, β-galactosidase, luciferase and the like as a fusion partner. These terms include polypeptides that have been post-translationally modified in a cell, for example, those that have undergone glycosylation, cleavage, secretion, prenylation, carboxylation, phosphorylation, etc., and polypeptides that have a secondary or tertiary structure. , Other moieties such as other polypeptides, atoms, cofactors, etc. that are strongly bound, eg, covalently or non-covalently, to a polypeptide.

  The term "complementary" as used herein refers to nucleotide sequences that base pair with a target nucleic acid of interest by hydrogen bonding. In standard Watson-Crick base pairing, adenine (A) base pairs with thymine (T) and guanine (G) base pairs with cytosine (C) in DNA. In RNA, thymine is replaced by uracil (U). Thus, A is complementary to T and G is complementary to C. Generally, "complementary" refers to nucleotide sequences that are completely complementary to a target of interest, such that each nucleotide in the sequence is complementary at a position corresponding to each nucleotide in the target nucleus. The nucleotide sequence is not perfectly complementary (100% complementary) to the non-target sequence, but the complementarity of the particular sequence of the nucleotide sequence with the non-target sequence still allows base pairing with the non-target sequence. ,% complementarity can be calculated to assess the likelihood of non-specific (non-target) binding. Generally, less than 50% complementarity does not result in non-specific binding. Also, under stringent hybridization conditions, less than 70% complementarity does not result in non-specific binding.

  The terms "ribonucleic acid" and "RNA" as used herein refer to a polymer composed of ribonucleotides.

  The terms “deoxyribonucleic acid” and “DNA” as used herein refer to a polymer composed of deoxyribonucleotides.

  The term "oligonucleotide" as used herein refers to a single-stranded nucleotide polymer of about 10-200 nucleotides in length, up to 300 or more nucleotides in length, for example up to 500 or more nucleotides in length. Oligonucleotides may be synthesized, and in some embodiments shorter than 300 nucleotides in length.

  The term "adhesion" as used herein refers to the strong, eg, covalent or non-covalent, connection of one molecule to another.

  As used herein, the term “surface adhesion” refers to the strong adhesion of molecules to a surface.

  The term “sample” as used herein relates to one or more analytes or entities-containing materials or mixtures of materials of interest. In certain embodiments, the sample can be obtained from biological samples such as cells, tissues, body fluids and faeces. The target body fluids are amniotic fluid, aqueous humor, vitreous humor, blood (eg, whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax, chyle, chyme, Endolymph, perilymph, feces, stomach acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, purulent fluid, mucous secretions, saliva, sebum (skin oil), semen , Sputum, sweat, synovial fluid, tears, vomiting, urine and exhaled breath condensate. In certain embodiments, the sample can be obtained from a subject, eg, a human, and can be processed prior to being used in a subject assay. For example, proteins/nucleic acids can be extracted from tissue samples prior to use, prior to analysis, and methods are known. In certain embodiments, the sample may be a clinical sample, eg, a sample taken from a patient.

  The term "analyte" refers to molecules having different shapes (eg proteins, peptides, DNA, RNA, nucleic acids or other molecules), cells, tissues, viruses and nanoparticles.

  The term "assay" refers to testing a sample to detect the presence and/or abundance of an analyte.

  As used herein, the terms "determination", "measurement" and "evaluation" are used interchangeably with "assay" and include quantitative and qualitative determinations.

  As used herein, the term "light-emittering label" refers to a label capable of emitting light under external excitation. It can emit light. Fluorescent labels (including dye molecules or quantum dots) and luminescent labels (eg electro or chemiluminescent labels) are types of fluorescent labels. External excitation refers to light (photons) for fluorescence, electric current for electroluminescence, and chemical reaction for chemiluminescence. External excitation may be a combination of the above.

  The phrase "labeled analyte" refers to an analyte that is detectably labeled with a luminescent label such that the analyte can be detected by assessing the presence of the label. The labeled analyte may be directly labeled (ie the analyte itself may be directly attached to the label via a strong bond, eg covalent or non-covalent), or the labeled analyte May be labeled indirectly (ie the analyte is directly bound to the labeled secondary capture agent).

  The terms "hybridization" and "conclusion" with respect to nucleic acids are used interchangeably.

  The term "capture agent/analyte complex" refers to the complex resulting from the specific binding of capture agent and analyte. Generally, the capture agent and the analyte for the capture agent bind specifically to each other under "specific binding conditions" or "conditions suitable for specific binding" and these conditions (salt concentration, pH, detergent, Protein concentration, temperature, etc.) are conditions that allow binding to occur between the capture agent and the analyte in solution. Those conditions, in particular for antibodies and their antigen and nucleic acid hybridizations, are well known in the art (eg Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, NY. ), Ausubel et al., Short Protocols in Molecular Biology, 5th Edition, Wiley & Sons, 2002).

  The terms "specific binding conditions" and "conditions suitable for specific binding" used for binding the capture agent herein to an analyte, for example, a biomarker, biomolecule, synthetic organic compound, inorganic compound, etc. Generate nucleic acid duplexes or protein/protein (eg, antibody/antigen) complexes, protein/compound complexes, aptamer/target complexes that include molecule pairs that specifically bind to each other, and Refers to conditions that are detrimental to the formation of complexes between unbound molecules. Specific binding conditions are the sum or combination (whole) of both hybridization and wash conditions and may include selection and blocking steps, if desired. For nucleic acid hybridization, 50% formamide, 5×SSC (150 mM NaCl, 15 mM sodium citrate), 50 mM sodium phosphate (pH 7.6), 5× Denhardt's solution, 10% dextran sulfate and 20 μg/ml denatured and sheared. Specific binding conditions can be achieved by incubating at 42° C. in a solution containing salmon sperm DNA, followed by washing the filters with 0.1×SSC at about 65° C.

  To bind the antibody to the antigen, the first plate containing the antibody is blocked with a blocking solution (eg, PBS containing 3% BSA or skim milk) followed by the analyte containing in diluted blocking buffer. Specific binding conditions are achieved by incubating with the sample. After incubation, the first plate is washed in wash solution (eg PBS+TWEEN 20) and incubated with a secondary capture antibody (detection antibody that recognizes a second site in the antigen). The secondary capture antibody may be conjugated to an optically detectable label, eg, a fluorophore such as IRDye 800CW, Alexa 790, Dylight 800. After further washing, the presence of bound secondary capture antibody can be detected. Those skilled in the art know that the parameters can be modified to increase the detected signal and reduce the background noise.

  The subject can be any human or non-human animal. The subject may be the person performing the method, the patient, the customer of the laboratory, etc.

  As used herein, an "analyte" is any substance suitable for testing the method.

  As used herein, "diagnostic sample" refers to any biological sample obtained from a subject, which is a bodily byproduct, such as a body fluid. The diagnostic sample can be obtained directly from the subject in the form of a liquid, or can be obtained from the subject by placing the body by-product in a solution, for example a buffer. Exemplary diagnostic samples include, but are not limited to, saliva, serum, blood, sputum, urine, sweat, tears, semen, feces, breath, biological tissue, mucus, and the like.

  As used herein, "environmental sample" refers to any sample obtained from the environment. Environmental samples include liquid samples from rivers, lakes, ponds, oceans, glaciers, icebergs, rain, snow, sewage, reservoirs, tap water, drinking water, soil, compost, sand, rocks, concrete, wood, bricks, It may include solid samples from sludge and the like, and gas samples from air, underwater heat vents, industrial exhaust gases, vehicle exhaust gases and the like. Generally, the sample in non-liquid form is converted to liquid form prior to analyzing the sample using the method of the invention.

  As used herein, a “food sample” refers to any sample suitable for animal consumption, eg human consumption. Food samples may include raw materials, cooked foods, animal and plant derived foods, pre-treated foods and partially or fully treated foods and the like. Generally, the sample in non-liquid form is converted to liquid form prior to analyzing the sample using the method of the invention.

  As used herein, the term "diagnosis" refers to the use of a method or analyte to identify, predict, and/or predict the therapeutic response of a disease or condition of interest. .. Diagnosis is predicting the likelihood or constitution of a disease or medical condition, estimating the severity of the disease or medical condition, determining the risk of progression of the disease or medical condition, and clinical response to treatment. And/or predicting response to treatment.

  As used herein, a "biomarker" is any molecule or compound discovered in a sample of interest that is diagnostic of the presence or constitution of the disease or condition of interest in a subject. , Or associated therewith, said sample being taken from a subject. Biomarkers include, but are not limited to, polypeptides or complexes thereof (eg, antigens, antibodies), nucleic acids (eg, DNA, miRNA, mRNA), drug metabolites, lipids, carbohydrates, hormones, vitamins, etc. It is known to be associated with the underlying disease or condition.

  As used herein, a "condition" associated with the diagnosis of a health condition refers to a physiological state of mind and body that can be distinguished from other physiological states. In some cases, the health condition may not be diagnosed with the disease. Exemplary health conditions of interest include nutritional health, aging, environmentally harmful substances, pesticides, herbicides, exposure to synthetic hormone analogs, pregnancy, menopause, male rest, sleep, stress, prediabetes, exercise. , Fatigue, chemical balance, etc., but not limited to these. The term "biotin moiety" refers to affinity reagents including biotin or biotin analogues such as desthiobiotin, oxybiotin, 2'-iminobiotin, diaminobiotin, biotin sulfoxide, biocytin and the like. The biotin moiety is bound to streptavidin with an affinity of at least 10-8M. The biotin affinity reagent may comprise a linker such as -LC-biotin, -LC-LC-biotin, -SLC-biotin, or PEGn-biotin where n is 3-12.

  The term "streptavidin" refers to streptavidin, avidin, and any variant thereof that binds biotin with high affinity.

  The term "marker" used to describe a biological sample refers to an analyte, the presence or abundance of which in the biological sample is associated with a disease or medical condition.

  The term "bond" includes hydrogen bonds, ionic bonds and covalent and non-covalent bonds, including van der Waal forces.

  The term "amplification" refers to the amplification of the magnitude of a signal, eg, at least 10-fold amplification, at least 100-fold amplification, at least 1,000-fold amplification, at least 10,000-fold amplification or at least 100-fold amplification in a signal. 1,000 times the amplification.

  The term “entity” refers to proteins, peptides, DNA, RNA, nucleic acids, molecules (small or large molecules), cells, tissues, viruses, nanoparticles of different shapes that bind to a “binding site”, Not limited. Entities include capture agents, detection agents and blockers. “Entity” includes “analyte” and these two terms are used interchangeably.

  The term "binding site" refers to a position on a solid surface that can immobilize a "entity" in a sample.

  The term "entity partner" means a protein, peptide, DNA, RNA, nucleic acid, molecule (small molecule or large molecule), cell, tissue, virus, nano-form of different shape, which is located at the "binding site" and binds to the entity. Refers to particles, but is not limited to these. Entities include, but are not limited to, capture agents, detection agents, secondary detection agents or "capture agents/analyte complexes".

  The term "target analyte" or "target entity" refers specifically to the particular analyte being analyzed (ie, detected), or the particular entity that specifically binds to the binding site.

  The terms “smartphone” or “cell phone”, used interchangeably, have a camera and communication hardware and software, use the camera to take images, process images taken by the camera, and remotely Refers to the type of mobile phone that can communicate data to the earth. In some embodiments, the smartphone has a flashlight.

  Unless otherwise stated, the term "light" refers to electromagnetic radiation having various wavelengths.

  The term "average linear dimension" of a region is defined as a length equal to the area of the region multiplied by 4 and divided by the perimeter of the region. For example, if the region is a rectangle, the width is W, and the length is L, the average linear dimension of the rectangle is 4*W*L/(2*(L+W)) (“* "Indicates multiplication and "/" indicates division). By this definition, each average linear dimension is W for a square of width W and d for a circle of diameter d. Regions include, but are not limited to, regions of binding or storage sites.

  The term "period" of a periodic structure array refers to the distance from the center of one structure to the center of the nearest adjacent same structure.

  The term "reservoir site" refers to the site of an area on a plate that contains a reagent that is added to the sample, and said reagent is capable of dissolving in the sample in contact with the reagent and diffusing into the sample. ..

  The term "relevant" relates to the detection of an analyte, the quantification and/or control of an analyte or entity in a sample or plate, or the quantification or control of a reagent added to a sample or plate. It means to do.

  The term “hydrophilic”, “wettable”, or “wettable” of a surface refers to the contact angle of the sample on the surface being less than 90 degrees.

  The term "hydrophobic", "non-wetting", or "non-wetting" of a surface refers to the contact angle of the sample on the surface of 90 degrees or more.

  The term "variation" with respect to quantity refers to the difference between the actual value and the target value or average value of the quantity. The term "relative variation" of an amount refers to the ratio of the variation value to a target value or average value of the amount. For example, if a certain amount of the target value is Q and the actual value is (Q+Δ), Δ is the variation value and Δ/(Q+Δ) is the relative variation value. The term "relative sample thickness variation" refers to the ratio of the sample thickness variation value to the average sample thickness.

  The term "light transmissive" refers to a property of a material that allows the transmission of optical signals, and unless otherwise specified, the term "optical signal" refers to a sample, plate, spacer, scale mark, or any used material. Refers to an optical signal used to detect properties of structures, or any combination thereof.

  The term "non-sample volume" refers to that in the closed configuration in the CROF process, the volume between the plates is occupied by other objects than the sample rather than the sample. These objects include, but are not limited to, spacers, bubbles, dust or any combination thereof. Generally, the non-sample volume is mixed inside the sample.

  The term "saturation incubation time" refers to the time required for the binding between two types of molecules (e.g. capture agent and analyte) to reach equilibrium. For surface-immobilized assays, "saturation incubation time" is the time required for the binding between the target analyte (entity) in the sample and the binding site on the plate surface to reach equilibrium, that is, depending on the binding site. Refers to the time after which the average number of target molecules (entities) captured and immobilized becomes statistically nearly constant.

  In some cases, "analyte", "binding entity" and "entity" are used interchangeably.

  "Processor", "communication device", "mobile device" is one of the basic electronic components (memory, input/output interface, central processing unit, instructions, network interface, power supply, etc.) for performing computational tasks. (Including the above). A computer system may be a general purpose computer or special purpose computer that contains instructions for performing a particular task.

  As used in describing signal or data communication, "site" or "location" refers to the local area in which the device or subject is located. A site refers to a small geographically defined area within a building structure, such as a room in a hospital, or a large geographically defined area. For a first site far away from the second site, the remote site or remote location is the first site physically separated from the second site by distance and/or physical obstacles. The remote site may be a first site away from a second site in the building structure, a first site in a different building structure from the second site, or a first site in a different city than the second site. May be.

  As used herein, the term "sampling site" refers to the location where a sample is obtained from a subject. The sampling location may be, for example, a retail store location (eg, chain store, pharmacy, supermarket or department store), provider's office, doctor's office, hospital, subject's home, military site, employer site or other site or It may be a combination of sites. As used herein, the term "sampling site" further refers to the owner or representative of a business, service or facility located at or affiliated with the site.

  As used herein, "raw data" includes signals and direct read data from sensors, cameras, other components and instruments that detect or measure properties or characteristics of a sample. For example, raw data may include voltage or current output from a sensor, detector, counter, camera, or other component or device, and raw data may include raw data from a sensor, detector, counter, camera, or other component or device. , Digital or analog numerical output, and raw data further includes digital or filtered outputs from sensors, detectors, counters, cameras or other components or devices. For example, raw data includes the output of a photometer, which includes the output in "relative light units" related to the number of photons detected by the photometer. Raw data may include JPEGs, bitmaps or other image files generated by the camera. Raw data includes cell number, light intensity (at or at a specific wavelength, or within or within a wavelength range), rate of change of detector output, and between two similar measurements. Difference, a plurality of detected events, the number of events detected within a preset range or satisfying preset criteria, and a minimum value measured within a certain period or in the visual field, The maximum value measured in a certain period or in the visual field and other data may be included. If the raw data is sufficient, it can be used without further processing or analysis. In other cases, the raw data may be further processed or used for sample, subject-related analysis or other purposes.

  "Representing a sample" as used in reference to an output signal or raw data representative of a sample is an output signal or raw data that reflects the measured property of the sample or a portion thereof, such as the amount of analyte present in the sample. Point to. For example, the intensity representing the fluorescence signal is higher in the fluorescence-labeled sample containing more analytes of interest than the fluorescence signal in the fluorescence-labeled sample containing less analyte.

  As used herein, "Clinical Laboratory Improvement Amendment Bill" and "CLIA" refer to 42 U.S.M. S. C. Part F clauses, such as subpart 2, paragraphs 263a-263a7, Federal Regulation 42 CFR Chapter W (493.1-493.2001), and related laws, regulations, and amendments. CLIA legislation is managed by the Medicare Medicaid Service Center (CMS) of the United States Department of Health and Human Services.

  As used herein, "process control" refers to any number of methods and systems for planning and/or monitoring the performance of processes such as sample analysis processes.

  As will be apparent to one of ordinary skill in the art upon reading this disclosure, each of the individual embodiments described and illustrated herein may be practiced in a number of other implementations without departing from the scope or spirit of the invention. It has individual components and features that are easily separated from or combined with any feature of the form. Any of the methods may be performed in the order of the events or any other order that is logically possible.

  Those skilled in the art will understand that the invention is not limited to the structural details, component placement, category selection, weighting, predetermined signal restrictions or application to steps described herein or illustrated in the drawings. Should be. The invention is capable of other embodiments and of being carried out or carried out in various ways.

  The practice of the various embodiments of the present disclosure, unless otherwise indicated, is within the skill of the art, as is conventional in immunology, biochemistry, chemistry, molecular biology, microbiology, cell biology, genomics and recombinant DNA. Use technology. Green and Sambrook, MOLECULAR CLONING: A LABORATORY MANUAL, 4th edition (2012), CURRENT PROTOCOLS IN MOLECULAR BIOLOGYc (FM) Uslovel et al. (1987), METHODS, METHODS. : A PRACTICAL APPROACH (MJ MacPherson, BD Hames and GR Taylor, edited (1995)), Harlow and Lane (1988), ANTIBODIES, A LABORATORY MANUAL, and UNICAL. See Freshney ed. (1987)).

  As used in this specification and the appended claims, the singular forms of "a", "a" and "a", unless the context clearly dictates otherwise, such as when using "single." It should be noted that "(an)" and "the" also include multiple meanings. For example, reference to "analyte" includes single and multiple analytes, reference to "capture agent" includes single capture agent and multiple capture agents, and reference to "detection agent". Then, it includes a single detection agent and a plurality of detection agents, and when referring to “reagent”, it includes a single reagent and a plurality of reagents.

Compression controlled open flow (CROF)
Manipulation of samples or reagents during the assay can improve the assay. The operations include, but are not limited to, manipulation of sample and/or reagent geometry and position, manipulation of sample or reagent mixing or binding, and manipulation of sample or reagent and plate contact area. Not limited.

  Many embodiments of the present invention use methods called "compressed controlled open flow (CROF)" and devices that perform CROF to manipulate sample and/or reagent geometric sizes, locations, contact areas and mixing. ..

  The term "compressed open flow (COF)" refers to (i) placing another plate on top of at least some of the samples, and (ii) then pressing the two plates against each other between the two plates. Method of changing the shape of a flowable sample adhered to a plate by compressing the sample, the compression reducing the thickness of at least a portion of the sample so that the sample opens between the plates. Flow into space.

  The term "compressed controlled open flow" or "CROF" (or "self-calibrating compressed open flow" or "SCOF" or "SCCOF") refers to a particular type of COF, which after compression is the final part of a sample or part of the sample. The thickness is "adjusted" by the spacer, which is placed between the two plates.

  In CROF, the term "the final thickness of some or all of the sample is adjusted by spacers" means that during CROF, when a particular sample thickness is reached, the relative movement of the two plates is stopped, This also stops the change in sample thickness, indicating that the particular thickness is determined by the spacer.

As shown in FIG. 1, one embodiment of the CROF method is
(A) obtaining a flowable sample,
(B) obtaining a first plate and a second plate that are movable relative to each other in different configurations, each plate having a substantially flat sample contact surface, One or two include spacers having a predetermined height and located on corresponding sample contact surfaces,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration comprising two plates partially or completely separated and between the plates. Wherein the spacing is not adjusted by the spacer, and (d)(c) the steps of spreading the sample in a closed configuration with the plates facing each other in the closed configuration. And a sample of relevant volume is located between the plates, the thickness of the sample of relevant volume is adjusted by the plate and the spacer, the relevant volume being at least part of the total volume of said sample, and The step of flowing the sample laterally between the two plates during the spread of the sample is included.

  Unless otherwise stated, the term “plate” refers to a plate used in a CROF process, which is a solid with a workable surface, that is, a sample placed between two plates and another plate. Compress with to reduce the thickness of the sample.

  The terms "the plate" or "the pair of the plate" refer to two plates in a CROF process.

  The term "first plate" or "second plate" refers to the plate used in the CROF process.

  The term "plates face each other" refers to the case where a pair of plates at least partially face each other.

  Unless otherwise stated, the term "spacer" or "stopper", when placed between two plates, sets a limit on the minimum spacing between the two plates that can be reached when the two plates compress together. Refers to a mechanical object. That is, in compression, the spacers stop the relative movement of the two plates so that the spacing between the plates does not become smaller than a preset value (ie a predetermined value). Spacers come in two types: "open spacers" and "closed spacers."

  The term "open spacer" refers to a spacer having a shape that allows liquid to flow around and around the spacer. For example, the post is an open spacer.

  The term "closed spacer" refers to a spacer having a shape such that liquid flows around the spacer and cannot flow through the spacer. For example, the ring-shaped spacer is a closed spacer for the liquid in the ring, and the liquid in the ring-shaped spacer stays in the ring and cannot go outside (outer periphery).

  The terms "spacer has a predetermined height" and "spacer has a predetermined distance between spacers" refer to the fact that the spacer height and the distance between spacers are known prior to the CROF process, respectively. . Prior to the CROF process, if the spacer height and the distance between spacers values are not known, they are not preset. For example, if the beads are sprinkled on the plate as spacers and the beads stop at random positions on the plate, the distance between the spacers is not preset. Another example where the distance between the spacers is not preset is that the spacers move in the CROF process.

  The term "spacers are fixed on their respective plates" in the CROF process means that the spacers are connected to a position on the plate and maintain the connection with that position during the CROF (ie the spacers on each plate). The position does not change). One example of "a spacer is fixed to its corresponding plate" is that the spacer is made integrally of plate material and that the spacer does not change position with respect to the plate surface during CROF. An example of "a spacer is not fixed to its corresponding plate" is that the spacer is attached to the plate with an adhesive, but when using the plate, the adhesive will cause the spacer to reposition itself on its plate surface during CROF. Instead of keeping it, the spacer moves away from its original position on the plate surface.

  The term "spacer is integrally fixed to the plate" means that the spacer and the plate do not move or leave their original position on the plate during use, like a monolithic object. Refers to.

  The term "open configuration" of two plates in a CROF process refers to a configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers.

  The term "closed configuration" of two plates in a CROF process means that the plates are facing each other, a spacer and an associated volume of sample are located between the plates, and the associated volume of sample thickness is Refers to the configuration adjusted by the spacer, the relevant volume being at least part of the total volume of the sample.

  The term "sample thickness is adjusted by plates and spacers" in the CROF process means that for a given condition of plate, sample, spacer and plate compression method, at least some of the samples in a closed configuration of the plate. It means that the thickness can be preset based on the characteristics of the spacer and the plate.

  The term "inner surface" or "sample surface" for a plate in a CROF device refers to the surface of the plate that contacts the sample, the other surface of the plate (which does not contact the sample) is called the "outer surface".

  The term "X-plate" of a CROF device refers to a plate including spacers on the sample surface of the plate, said spacers having a predetermined inter-spacer distance and spacer height, and at least one spacer being Within the sample contact area.

  The term "CROF device" refers to a device that performs a CROF process. The term "performing CROF" refers to using the CROF process. For example, the term "perform CROF on a sample" refers to placing the sample in a CROF device and performing the CROF process, which holds the sample in its final configuration in CROF unless otherwise specified.

  The term "CROF plate" refers to two plates used to carry out a CROF process.

  The term "surface smoothness" or "surface smoothness variation" for a flat surface refers to the average deviation between a flat surface and a perfectly flat surface over a short distance of a few micrometers or less. Surface smoothness differs from surface flatness variation. A flat surface has good surface flatness, but its surface smoothness is low.

  The term "surface flatness" or "surface flatness variation" for a flat surface refers to the average deviation between a flat surface and a perfectly flat surface over a long distance of about 10 μm or more. Surface flatness variation is different than surface smoothness. A flat surface has good surface smoothness, but its surface flatness is low (ie its surface flatness variation is large).

  The term "relative surface flatness" for a plate or sample refers to the ratio of plate surface flatness variation to the final sample thickness.

  Unless otherwise stated, the term "final sample thickness" in the CROF process refers to the sample thickness in the closed configuration of the plate during the CROF process.

  The term "compression method" in CROF refers to the method of moving two plates from an open configuration to a closed configuration.

  The term "region of interest" or "region of interest" for a plate refers to the region of the plate that is associated with the function performed by the plate.

  The term "maximum" refers to "below". For example, the height of the spacer is 1 μm at maximum, which means that the height of the spacer is 1 μm or less.

  The term "sample area" refers to the area in the direction in which the sample is approximately parallel to the space between the plates and perpendicular to the thickness of the sample.

  The term "sample thickness" refers to the sample dimension in a direction perpendicular to the surfaces of the plates facing each other (eg, the direction of the distance between the plates).

  The term "distance between plates" refers to the distance between the inner surfaces of two plates.

  The term "final sample thickness deviation" in CROF refers to the difference between a given spacer height (determined by spacer manufacture) and the average final sample thickness, The average final sample thickness is the average value for a given area (eg, the average value of 25 different points (distance is 4 mm) in a 1.6 cm×1.6 cm area).

  The term "uniformity of measured final sample thickness" in the CROF process refers to the standard deviation (eg, standard deviation from the mean) of the final sample thickness measured in a given sample area.

  The terms "sample associated volume" and "sample associated area" in the CROF process respectively refer to the volume and area of some or all of the sample deposited on the plate during the CROF process, depending on the respective method or Related to the function performed by the device, this function is to reduce the binding time of the analyte or entity, detect the analyte, quantify the volume, quantify the concentration, mix the reagents, or the concentration (analyte, entity. , Or reagent) control, but is not limited to these.

  Unless otherwise indicated, the terms “some embodiments”, “in some embodiments”, “in some embodiments of the present invention”, “embodiment”, “one embodiment”, “others”. “Embodiment”, “specific embodiment”, “many embodiments”, etc. are embodiments applied to all disclosures (that is, all inventions).

  Unless otherwise indicated, the term "height" or "thickness" of an object in the CROF process refers to the dimension of the object in a direction perpendicular to the surface of the plate. For example, the height of the spacer is the dimension of the spacer in the direction perpendicular to the surface of the plate, and the height of the spacer and the thickness of the spacer are the same.

  Unless otherwise indicated, the term "area" of interest in a CROF process refers to the area of interest parallel to the surface of the plate. For example, the area of the spacer refers to the area of the spacer parallel to the surface of the plate.

  Unless otherwise indicated, the term "lateral" or "laterally" in the CROF process refers to the direction parallel to the surface of the plate.

  Unless otherwise indicated, the term "width" in the CROF process refers to the lateral dimension of the spacer.

  The term “spacer within the sample” refers to the spacer being surrounded by the sample (eg, a columnar spacer within the sample).

  The term "critical bending span" of a plate in the CROF process refers to the span (ie, distance) of the plate between two supports, and for a given flexible plate, sample, and pressure, plate bending is acceptable. Equal to a simple curve. For example, for a given flexible plate, sample and pressure, if the acceptable curvature is 50 nm and the critical curvature span is 40 μm, the plate curvature between two adjacent spacers 40 μm apart is 50 nm. , And two adjacent spacers smaller than 40 μm, the curvature is smaller than 50 nm.

  The term "flowable" with respect to a sample refers to an increase in its lateral dimension as the sample thickness decreases. For example, a faecal sample would be considered flowable.

  In some embodiments of the invention, as long as the sample thickness can be reduced in the CROF process, the sample in the CROF process does not need to be flowable and thus benefits from the process. For example, placing the dye on the surface of a CROF plate to stain the tissue and reducing the thickness of the tissue during the CROF process can speed up the saturation incubation time for staining with the dye.

1. Reduction (shortening) of binding or mixing time (X)
Reduced incubation/reaction times for performing assays or other chemical reactions are desirable. For example, in a surface-immobilized assay that captures a target analyte in a sample with a capture agent (ie, solid phase) immobilized on the plate surface, the saturation incubation time for capturing the target analyte in the sample is generally It is desirable to have a short time, or a short time to immobilize the capture and detection agents in solution on the plate surface, or both. Another example is the need to reduce the time to apply the capture agent to the plate surface. Another example is the need to reduce the mixing time of reagents and samples.

  The present invention provides methods and devices that reduce (ie, shorten) the saturation incubation time (ie, volume-to-surface entity time) required to bind an entity in a sample to a binding site on a solid surface. Another aspect of the invention reduces the time required to bind an entity stored on the surface of a plate to a binding site on the surface of another plate (ie, the time of an entity from one surface to another). That is. Another aspect of the invention reduces the time required to add/mix a surface-stored reagent to a fixed volume of sample (ie, time of reagent added/mixed from the surface to a fixed volume of sample). That is.

  The present invention reduces the saturation incubation time of binding and/or mixing in an assay by using an apparatus and method that spreads the sample (or liquid) to a thin thickness, thereby reducing the diffusion time of the entity over the sample thickness. .. The diffusion time of an entity in a material (eg, liquid or solid or semi-solid) is directly proportional to the square of the diffusion distance, thus decreasing the sample thickness can reduce the diffusion distance, and the diffusion time and saturation incubation time can be dramatically reduced. Can cause a decrease. The small thickness (eg, a narrow enclosed space) also increases the frequency of collisions of the entity with other entities in the material, thereby further enhancing binding and mixing. The method according to the invention is further applied to make the reduction of sample thickness accurate, uniform, fast, simple (with fewer operating steps) and to reduce the sample thickness to micrometer or nanometer thickness. It These inventions are rapid, low cost and have high utility in terms of PoC, diagnostics and chemical/biological analysis. 1-4 show some embodiments of the present invention.

1.1 Reduction of Saturation Incubation Time for Binding of Entities in Sample to Binding Sites on Solid Surface by Decreasing Sample Thickness X1. A method for reducing the saturation incubation time required to bind a target entity in a sample to a binding site on a plate surface, as shown in Figures 1-2, 3a and 4a, comprising:
(A) obtaining a sample containing a target entity that is flowable and can diffuse into the sample;
(B) obtaining a first plate and a second plate movable relative to each other in different configurations, the first plate being configured to bind to a target entity on its surface. One or two of said plates having bound binding sites and comprising spacers fixed to their corresponding plates and having a predetermined height,
(C) depositing the sample on one or two plates when the plates are configured in an open configuration, the open configuration including two plates partially or completely separated and between the plates. The spacing is not adjusted by the spacer, the configuration is a step,
(D) after (c) spreading the samples in a closed configuration with the plates in the closed configuration with the plates facing each other and the spacer and associated volume of the sample located between the plates The site is in contact with the relevant volume and the thickness of the relevant volume of the sample is adjusted by the plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration,
The relevant volume is a portion or the total volume of the sample, and the reduced sample thickness reduces the saturation incubation time for binding the target entity in the relevant volume to the binding site. Including a method.

  For a given sample volume, CROF reduces the sample thickness but increases the lateral dimension of the sample. The present invention uses the above facts to perform (a) local binding or mixing in a portion of the sample, and (b) multiplex binding or mixing sites in the absence of a fluid barrier. , Separate the sample fluid into different isolation bags.

X2. An apparatus for reducing the saturation incubation time required to bind a target entity in a sample of relevant volume to a surface, as shown in Figures 1-2, 3a and 4a,
(A) moveable relative to each other in different configurations; (b) each plate has a sample contact area for contacting the sample, the sample being a target entity in an associated volume of sample. And (c) one of the plates has a binding site for binding a target entity, and (d) at least one of the plates comprises a spacer, the spacer having a predetermined distance and height between the spacers. And fixed to its respective surface, at least one of the spacers being located in the sample contact area, including a first plate and a second plate,
One configuration is an open configuration in which the two plates are partially or completely separated and the spacing between the plates is not adjusted by spacers,
Another configuration is the closed configuration, which was configured after depositing the sample in the open configuration, in which the plates face each other and the spacer and associated volume of sample are located between the plates and the binding site. Is in contact with the relevant volume and the thickness of the relevant volume of the sample is adjusted by the plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration. The volume of the sample is a partial or total volume of the sample, and the reduced sample thickness reduces the saturation incubation time required to bind the target entity to the binding site in the volume of interest.

1.2 Decrease in saturation incubation time required to bind a substance stored on the surface of one plate to a binding site on the surface of another plate X3. Storage of one plate, as shown in Figures 1, 3c and 4b A method for reducing the saturation incubation time for binding a site-stored entity to a relevant binding site on another plate, comprising:
(A) Obtaining a first plate and a second plate that are movable relative to each other in different configurations, the surface of the first plate having a binding site and the second plate The plate has a reservoir containing an entity that binds to a binding site, the area of the binding site and the area of the reservoir are smaller than the area of its corresponding plate, and one or two of the plates comprises spacers. , Each of the spacers being fixed to its corresponding plate and having a predetermined height, a step,
(B) obtaining the transport medium, wherein the entity at the storage site is capable of dissolving and diffusing in the transport medium,
(C) applying a transfer medium to one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated. A configuration in which the spacing between the plates is not adjusted by spacers, steps,
(D) After (c), the steps of spreading the sample in the closed configuration of the plates, wherein the plates face each other and the spacers, binding sites, storage sites and at least some of the transfer medium are in the plate. And the binding site at least partially overlaps the storage site, the transport medium contacts at least a portion of the binding site and the storage site, and the thickness of the transport medium is adjusted by the plates and spacers. Including a step of less than a maximum thickness of the transfer medium when the plate is in the open configuration,
The method wherein the reduced thickness of the transfer medium reduces the time required to bind the entities stored in the second plate to the binding sites on the first plate.

  Explain the reason. Includes tissue staining. A target entity that diffuses vertically across the sample thickness is described.

X4. An apparatus for reducing the saturation incubation time for binding an entity stored in a storage site of one plate to a binding site on another plate, as shown in Figures 1, 3c and 4b, comprising:
Movable relative to each other in different configurations, the first plate has a binding site on its surface, and the second plate has a storage site on its surface containing an entity that binds to the binding site. And the area of the binding site and the area of the storage site is less than the area of its respective plate, and one or two of said plates comprises spacers, each of the spacers being fixed to its respective plate and and Including a first plate and a second plate having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the transfer medium adheres to one or two of said plates, The above entity can dissolve in the transport medium and diffuse into the transport medium,
Another configuration is a closed configuration that is configured after the transfer medium is deposited in the open configuration, wherein the plates face each other and the spacers, binding sites, storage sites, and at least some of the transfer medium are in the plate. And the binding site at least partially overlaps the storage site, the transport medium contacts at least a portion of the binding site and the storage site, and the thickness of the transport medium is adjusted by the plates and spacers. , Less than the maximum thickness of the transfer medium when the plate is in the open configuration,
An apparatus that reduces the saturation incubation time required to bind entities on the storage site of the second plate to binding sites on the first plate due to the reduced thickness of the transfer medium.

  In some embodiments, in the method of paragraph X3 and the apparatus of paragraph X4, the transfer medium comprises a liquid in which the entities or reagents or both diffuse.

  In some embodiments, in the method of paragraph X3 and the apparatus of paragraph X4, the transfer medium is a sample containing an analyte (also referred to as a target analyte) that binds to the binding site.

  In some embodiments, in the method of Paragraph X3 and the apparatus of Paragraph X4, the transfer medium is a sample containing an analyte (also referred to as a target analyte) that binds to the binding site, and the sample binds the analyte. It is a detection agent.

1.3 Reduced time to add (mix) surface-stored reagents to liquid samples Many assays require the addition of reagents to the sample (including liquid). Generally, it is necessary to control the concentration of the reagent added to the sample or liquid. What is needed is a new method for performing such reagent addition and concentration control that is simple and/or low cost. Two examples of desired reagent additions are (a) hemocytometry, where anticoagulants and/or staining reagents are added to blood samples, and (b) immunology where detectors are added to bind target analytes in solution. It is a dynamic assay.

  One aspect of the present invention is a method, apparatus, and system that makes such reagent addition and concentration control simple and/or low cost. In one embodiment of the invention, a reagent layer (eg, a dried reagent layer) is first placed on the plate surface of a CROF device, then the sample is attached to the CROF device and the sample is contacted with the reagent in a CROF process. And make the sample thickness less than the sample thickness when the CROF plate is in the open configuration. By reducing the sample thickness, the diffusion time from the surface of the reagent to the entire sample is reduced, thereby reducing the time for reagent and sample mixing.

X5. A method for reducing the time to mix a reagent stored on a plate surface with a sample, as shown in Figures 1, 3b and 4c, comprising:
(A) a step of obtaining a first plate and a second plate that are movable with respect to each other so as to have different configurations, the first plate including a reagent added to a sample on its surface Has a storage site, the reagent is soluble in the sample and can diffuse into the sample, one or two of the plates comprises spacers, and each spacer is fixed on its respective plate and has a predetermined height. Having a step,
(B) obtaining a sample,
(C) depositing the sample on one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) After (c), the plate is opened in the closed configuration to spread the sample, and in the closed configuration, the plates face each other and the spacer, the storage site and at least a portion of the sample are located between the plates, Contacting at least a portion of the storage site, the thickness of the sample on the storage site being adjusted by plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration,
The method wherein the reduced thickness of the sample reduces the time to mix the sample with the reagent on the storage site.

  In the method of paragraph X5, it further comprises the step of incubating while the plate is in the closed configuration, wherein a large amount of reagent dissolved in the sample is included in the relevant volume of the sample at the selected incubation time. Thus, the relevant volume refers to the volume of the sample located at the storage site, and the incubation refers to the process that allows the reagents to dissolve and diffuse into the sample.

  The method of paragraph X5 provides the time after step (d) the time during which the plate is in the closed configuration, the time during which the agent is diffused in the sample over the thickness of the sample controlled by the plate while in the closed configuration. Incubating for the following times and then terminating the incubation further comprises allowing the reagents to diffuse into the sample, wherein the factors 0.0001, 0.001, 0.01, 0. 1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000 or any range between these values. For example, if the factor is 1.1 and the diffusion time is 20 seconds, the incubation time is 22 seconds or less. In a preferred embodiment, the factor is 0.1, 1, 1.5 or any range between these values.

X6. As shown in FIGS. 1, 3b and 4c, a device for reducing the time for adding a reagent stored on the plate surface into a sample,
Movable relative to each other in different configurations, the first plate has on its surface a storage site containing a reagent to be added to the sample, the reagent being soluble in the sample and A first plate and a second plate that are diffusable, one or two of said plates comprising spacers, and each spacer being fixed at its respective plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration configured after the transfer medium is deposited in the open configuration, wherein the plates face each other and the spacer, the storage site and at least a portion of the sample are located between the plates. The sample is in contact with at least a portion of the reservoir and the thickness of the sample on the reservoir is adjusted by the plate and spacer, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration,
An apparatus in which the reduced thickness of the sample reduces the time to mix the sample with the reagent on the storage site.

  The method or apparatus of any of paragraphs X1-6, in some embodiments, the relevant volume of sample is located at the binding site or storage site (ie, at the top of it). Is the volume of.

  In the method or apparatus of any of paragraphs X1-6, in some embodiments, the relevant volume of the sample is located in all or part of the binding site or storage site (ie. , Located on top of it) is the volume of the sample.

  The method or apparatus of any one of paragraphs X1-6, in some embodiments, the ratio of the lateral dimension of the binding site or storage site to the sample thickness in the closed configuration is 1.53 or greater, 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 1000 or more, 10,000 or more, or a range between any two of these values.

  The method or apparatus of any of paragraphs X1-6, wherein the ratio of the lateral dimension of the binding site or storage site to sample thickness in the closed configuration is between 3-20 in preferred embodiments, and In another preferred embodiment, between 100 and 1000, and in another preferred embodiment, between 1000 and 10,000.

  The method of any one of paragraphs X1 and X3, wherein in some embodiments the final reduced sample thickness is significantly less than the sample thickness over the area of the binding site, thereby A longer time is required to bind the entity in the outer sample area to the binding site. In the selection of the appropriate incubation time, the entity bound to the binding site is the entity in the sample volume located primarily at the binding site (ie the sample volume just above the binding region). Next, the calculation of the substance concentration in the sample is based on the sample thickness and the joint area.

  In the method of paragraph X5, in some embodiments, the final reduced sample thickness is significantly less than the sample thickness on the area of the storage site, thereby creating an entity in the sample region located outside the binding site. Requires longer time to bind to the binding site.

  With the selection of an appropriate incubation time, the entity bound to the binding site is the entity in the sample volume located primarily at the binding site (ie the sample volume just above the binding region). Next, the calculation of the substance concentration in the sample is based on the sample thickness and the joint area.

  The method of any one of paragraphs X2, X4, X6 further comprising a compression device for moving the plate from the open configuration to the closed configuration. In some embodiments, the compression device is one or any combination of the embodiments described in this disclosure.

  The method of any one of paragraphs X2, X4, X6, including a compression device for moving the plate from the open configuration to the closed configuration, and a retaining device configured to retain the plate in the closed configuration. In some embodiments, the retention device is one or any combination of the embodiments described in this disclosure.

  A method according to any of paragraphs X2, X4, X6, wherein a compression device is used to change the plate from an open configuration to a closed configuration, 0.001 sec or less, 0.01 sec or less, 0.1 sec or less, 1 sec. 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 10 minutes or less, 20 minutes or less, 30 minutes or less, A retention device configured to retain the plate in the closed configuration for 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less, 250 minutes or less, or a time in between any two of these values. Including and

  A method according to any of paragraphs X2, X4, X6, wherein the compression device changes the plate from an open configuration to a closed configuration, and in a preferred embodiment 0.001 sec or less, 0.01 sec or less, 0.1 Seconds or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, or between any two of these values A holding device configured to hold the plate in the closed configuration for a time within the range.

  Final Sample Thickness Final sample thickness in the closed configuration of the plate can be an important factor in reducing saturation incubation time. As discussed with respect to the adjusted spacing of the plates, the final sample thickness after sample thickness reduction/deformation depends on the nature and nature of the sample and the application.

  In some embodiments, the final sample thickness is less than about 0.5 μm (micrometer), less than about 1 μm, less than about 1.5 μm, less than about 2 μm, less than about 4 μm, about 6 μm. Less than about 8 μm, less than about 10 μm, less than about 12 μm, less than about 14 μm, less than about 16 μm, less than about 18 μm, less than about 20 μm, less than about 25 μm, less than about 30 μm, about 35 μm Less than, less than about 40 μm, less than about 45 μm, less than about 50 μm, less than about 55 μm, less than about 60 μm, less than about 70 μm, less than about 80 μm, less than about 90 μm, less than about 100 μm, about 110 μm Less than, less than about 120 μm, less than about 140 μm, less than about 160 μm, less than about 180 μm, less than about 200 μm, less than about 250 μm, less than about 300 μm, less than about 350 μm, less than about 400 μm, about 450 μm Smaller than about 500 μm, smaller than about 550 μm, smaller than about 600 μm, smaller than about 650 μm, smaller than about 700 μm, smaller than about 800 μm, smaller than about 900 μm, smaller than about 1000 μm (1 mm), about 1.5 mm Less than, less than about 2 mm, less than about 2.5 mm, less than about 3 mm, less than about 3.5 mm, less than about 4 mm, less than about 5 mm, less than about 6 mm, less than about 7 mm, less than about 8 mm Small, less than about 9 mm, less than about 10 mm, or a range between any two of these values.

  In certain embodiments, the final sample thickness in the closed configuration is less than 0.5 μm (micrometer), less than 1 μm, less than 5 μm, less than 10 μm, less than 20 μm, less than 30 μm, less than 50 μm. Less than 100 μm, less than 200 μm, less than 300 μm, less than 500 μm, less than 800 μm, less than 200 μm, less than 1 mm (millimeter), less than 2 mm (millimeter), less than 4 mm (millimeter), 8 mm (millimeter) It is less than or a range between any two of these values.

  In certain embodiments, the Q method makes the final sample thickness uniform and uses flat surfaces of the first and second plates.

  In the present invention, sample incubations are performed with and without shaking at various temperatures, humidities, gaseous environments and for various durations.

  Incubation time The method according to any of paragraphs X1 and X3 is carried out after the step (d) in the sample over the thickness of the sample adjusted by the plate in the closed configuration while the plate is in the closed configuration. Further comprising the step of incubating for a period of time less than or equal to the time the factor is applied to the time of diffusion, and then terminating the incubation, said incubation allowing the entity to bind to the binding site, said factor being 0.0001, 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000 or these Any range between values. For example, if the factor is 1.1 and the diffusion time is 20 seconds, the incubation time is 22 seconds or less. In one preferred embodiment, the factor is 0.1, 1, 1.5 or any range between these values.

  The method according to any one of paragraphs X5 comprises, after step (d), the entire sample over the thickness of the sample being adjusted by the plate while the entity is in the closed configuration while the plate is in the closed configuration. Incubating for a period of time less than or equal to the time the factor is diffused and then terminating the incubation, wherein said incubation allows the entity to bind to the binding site, said factor 0.0001,0 0.001, 0.01, 0.1, 1, 1.1, 1.2, 1.3, 1.5, 2, 3, 4, 5, 10, 100, 1000, 10,000 or a value thereof Any range between. For example, if the factor is 1.1 and the diffusion time is 20 seconds, the incubation time is 22 seconds or less. In one preferred embodiment, the factor is 0.1, 1, 1.5 or any range between these values.

  In the method described in any of paragraphs X1, X3, and X5 or the apparatus described in any of paragraphs X2, X4, and X6, at least one spacer is located within the sample contact region.

  In the method of any of paragraphs X1, X3, and X5 or the apparatus of any of paragraphs X2, X4, and X6, the spacers have a predetermined spacer distance.

  The method of any of paragraphs X1, X3, X5, further comprising an incubation step while the plate is in the open configuration, wherein the saturation incubation time is 0.001 seconds or less, 0.01 seconds or less, 0. 1 second or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 10 minutes or less, 20 minutes Hereafter, it is 30 minutes or less, 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less, 250 minutes or less, or a range between any two of these values.

  The method of any of Paragraphs X1, X3, X5, wherein the saturation incubation time at a reduced sample thickness in the closed configuration is 0.001 seconds or less, 0.01 seconds or less, 0.1 seconds or less, 1 second or less, 5 seconds or less, 10 seconds or less, 20 seconds or less, 30 seconds or less, 40 seconds or less, 1 minute or less, 2 minutes or less, 3 minutes or less, 5 minutes or less, 10 minutes or less, 20 minutes or less, 30 minutes or less Hereafter, it is 60 minutes or less, 90 minutes or less, 120 minutes or less, 180 minutes or less, 250 minutes or less, or a range between any two of these values.

  In some embodiments, the capture agent is first immobilized at the binding site, then the sample is contacted with the binding site, the entity in the sample is captured by the capture agent, and finally the detection agent is added to capture the captured entity. Bind to and read the signal from the detection agent (eg, by optical or electronic methods or combinations). In some embodiments, capture agents and other reagents besides detection agents (eg, blocking agents) are added.

  In many PoC applications, for example, it is desirable to have a simple and/or low cost device and method for adding additional reagents to a sample. One aspect of the present invention relates to a simple and/or low cost device and method for adding additional reagents to a sample. Additional reagents added include detection agents, blockers, light signal enhancers, light signal quenchers or other reagents. In some embodiments of the invention, different release times of reagents stored at the same location control the assay process. Different release times can be tailored by adding other materials with different dissolution rates.

  In certain embodiments, controlling the sample thickness (eg, controlling the ratio of sample thickness to storage site area and/or mixing time) controls the concentration of reagent mixed in the sample.

2. Plates, Spacers, Scale Markers, Adjusting Sample Thickness 2.1 Adjusting Plate Configuration and Sample Thickness Open Configuration In some embodiments, the open configuration provides two plates (ie, a first plate and a first plate and a second plate). The two plates) are separated from each other. In a particular embodiment, the two plates have one side connected together during all operating periods of the plate (including open and closed configurations), and the opening and closing of the two plates is Similar to. In some embodiments, the two plates have a rectangular (or square) shape with two rectangular sides connected during all operating periods of the plate.

  In some embodiments, the open configuration includes features in which the plates are far apart from each other such that the sample adheres to one of the pair of plates without interference with the other pair of plates.

  In some embodiments, the open configuration includes features where the plates are far apart from each other so that the sample is attached directly to one plate so that another plate is not present.

  In some embodiments, the open configuration has a distance between the pair of plates of at least 10 nm, at least 100 nm, at least 1000 nm, at least 0.01 cm, at least 0.1 cm, at least 0.5 cm, at least 1 cm, at least 2 cm, Or at least 5 cm, or any two value ranges.

  In some embodiments, the open configuration includes features in which the pair of plates are oriented in different directions. In some embodiments, the open configuration includes a configuration that defines an access gap between a pair of plates configured to allow sample addition.

  In some embodiments, the open configuration includes a configuration in which each plate has a sample contact surface and at least one contact surface of the plate is exposed when the plate is in the open configuration.

  Closing Configuration and Adjusting Sample Thickness In the present invention, a two plate closing configuration is one in which the spacing (ie distance) between the inner surfaces of the two plates is adjusted by a spacer between the two plates. The inner surface of the plate (also referred to as the "sample surface") contacts the sample during the compression step of the CROF process, thus in the closed configuration the sample thickness is adjusted by the spacer.

  During the process of moving the plates from the open configuration to the closed configuration, the plates face each other (at least some of the plates face each other) and force forces the two plates together. When the two plates are moved from the open configuration to the closed configuration, the inner surfaces of the two plates compress the sample attached to the plates to reduce the sample thickness (at the same time the sample is opened laterally between the plates). Flow), the thickness of the relevant volume of the sample is determined by the spacer, the plate, and the method used, and the mechanical/fluid properties of the sample. For a given sample and a given spacer, plate and plate press method, the thickness in the closed configuration can be predetermined.

  The terms "adjusting the spacing between the inner surfaces of the plates with spacers" or "adjusting the sample thickness with plates and spacers" or "the sample thickness is adjusted with spacers and plates" mean that the sample thickness in the CROF process is predetermined. It is determined by the plate, spacer, sample, and pressing method.

  In some embodiments, the sample thickness adjusted in the closed configuration is the same as the height of the spacer, in which case the spacer is in direct contact with the two plates (one plate The plate on which the spacer is fixed, and the other plate is the plate that contacts the spacer).

  In a particular embodiment, the sample thickness adjusted in the closed configuration is greater than the height of the spacer, in which case in the closed configuration the spacer directly contacts only the plate having the spacer fixed or attached to its surface. , Indirectly contacting another plate (ie, indirect contact). The term "indirect contact with" the plate means that the spacer and the plate are separated by a thin sample layer called the "residual sample layer", the thickness of which is referred to as the "residual thickness". be called. For a given spacer and plate, a given plate pressing method and a given sample, the thickness of the residue is preset (presetting means before the closed configuration), and the sample thickness is preset in the closed configuration. Set. This is because for a given condition (sample, spacer, plate and pressure) the thickness of the residual layer is the same and can be pre-calibrated and/or calculated. The adjusted sample thickness is approximately equal to the spacer height plus the thickness of the sample residue.

  In many embodiments, the post size and shape are pre-characterized (ie, preset) prior to use. The preset information is used in subsequent assays such as determination of sample volume (or relevant volume) and others.

  In some embodiments, adjusting the sample thickness comprises applying a closing (compressing) force to the plates to maintain the distance between the plates.

  In some embodiments, adjusting the sample thickness includes establishing a distance between the plates by a spacer, a closing force applied to the plates, a physical property of the sample, and the physical property of the sample is preferably Includes viscosity and compressibility.

2.2 Plate In the present invention, in general, a plate of CROF can be used (i) together with a spacer to control the thickness of part or all of the volume of the sample, and (ii) the sample to be assayed. Or made of a material that does not have a significant adverse effect on the goals the plate is trying to achieve. However, in certain embodiments, a particular material (and thus its properties) is used in the plate to achieve a particular purpose.

  In some embodiments, the two plates are for each parameter of plate material, plate thickness, plate shape, plate area, plate flexibility, plate surface characteristics and plate light transmission. Have the same or different parameters.

  Plate Materials Plates are made of single materials, composite materials, multiple materials, multiple layers of materials, alloys or combinations thereof. Each material used for the plate is an inorganic material, an organic material or a mixture, examples of the materials are described in the paragraphs Mat-1 and Mat-2.

  Mat-1. Inorganic materials used for the plates include glass, quartz, oxides, silicon dioxide, silicon nitride, hafnium oxide (HfO), aluminum oxide (AIO), semiconductors (silicon, GaAs, GaN, etc.), metals (eg gold, silver). , Copper, aluminum, titanium, nickel, etc.), ceramics or any combination thereof.

  Organic materials used for Mat-2 spacers include, but are not limited to, polymers (eg, plastics) or amorphous organic materials. The polymer material used for the spacer is an acrylate polymer, a vinyl polymer, an olefin polymer, a cellulosic polymer, a non-cellulosic polymer, a polyester polymer, nylon, a cyclic olefin copolymer (COC), poly(methyl methacrylate) (PMMA), polycarbonate (PC), Cyclic olefin polymer (COP), liquid crystal polymer (LCP), polyamide (PA), polyethylene (PE), polyimide (PI), polypropylene (PP), poly(phenylene ether) (PPE), polystyrene (PS), polyoxymethylene (POM), polyether ether ketone (PEEK), polyether sulfone (PES), poly(ethylene phthalate) (PET), polytetrafluoroethylene (PTFE), polyvinyl chloride (PVC), polyvinylidene fluoride (PVDF), Include, but are not limited to, polybutylene terephthalate (PBT), fluorinated ethylene propylene (FEP), perfluoroalkoxy alkanes (PFA), polydimethyl siloxane (PDMS), rubber, or any combination thereof.

  In some embodiments, the plates are each made of at least one of glass, plastic, ceramic and metal. In some embodiments, the plates each include at least one of glass, plastic, ceramic and metal.

  In some embodiments, one plate differs from other plates in side area, thickness, shape, material, or surface treatment. In some embodiments, one plate is the same as the other plate in terms of side area, thickness, shape, material, or surface treatment.

  The material used for the spacer is rigid, flexible, or any flexible in between. Rigidity (ie, hardness) or flexibility is related to the pressure used in placing the plate in the closed configuration.

  In some embodiments, the choice of rigid or flexible plate is determined from the requirements of controlling sample thickness uniformity in the closed configuration.

  In some embodiments, at least one of the two plates (for light) is transparent. In some embodiments, at least a portion or portions of one plate or two plates are transparent. In some embodiments, the plate is non-transparent.

  Plate Thickness In some embodiments, the average thickness of at least one plate is 2 nm or less, 10 nm or less, 100 nm or less, 500 nm or less, 1000 nm or less, 2 μm (micrometer) or less, 5 μm or less, 10 μm or less, 20 μm. Hereinafter, it is 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm (millimeter) or less, 2 mm or less, 3 mm or less, or a range between any two of these values.

  In some embodiments, at least one plate has an average thickness of up to 3 mm (millimeters), up to 5 mm, up to 10 mm, up to 20 mm, up to 50 mm, up to 100 mm, up to 500 mm, or any two of these values. It is the range between.

  In some embodiments, the plate thickness is not uniform across the plate. Different plate thicknesses at different locations can be used to control plate curvature, folding, sample thickness adjustment, and the like.

  Plate Shape and Area In general, the plate can have any shape so long as the shape allows for controlled open flow of the sample and sample thickness. However, in certain embodiments, certain shapes may be advantageous. The shape of the plates may be circular, oval, rectangular, triangular, polygonal, annular or any overlap of these shapes.

In some embodiments, the two plates may have the same or different sizes or shapes. The area of the plate depends on its application. The area of the plate is maximum 1 mm ² (square millimeter), maximum 10 mm ² , maximum 100 mm ² , maximum 1 cm ² (square cm), maximum 5 cm ² , maximum 10 cm ² , maximum 100 cm ² , maximum 500 cm ² , maximum 1000 cm ² , maximum 5000 cm. ² , up to 10,000 cm ² , or 10,000 cm ² or more, or a range between any two of these values. The shape of the plate may be rectangular, square, circular, or other shape.

  In certain embodiments, at least one of the plates is in the form of a belt (or strip) having a width, thickness, and length. Width is 0.1 cm (centimeter) maximum, 0.5 cm maximum, 1 cm maximum, 5 cm maximum, 10 cm maximum, 50 cm maximum, 100 cm maximum, 500 cm maximum, 1000 cm maximum, or a range between any two of these values. Is. The length can be as long as desired. The belt can be wound in a roll.

  Plate Surface Flatness In many embodiments, the inner surface of the plate is a flat or very flat surface. In certain embodiments, the two inner surfaces are parallel to each other in the closed configuration. The flat inner surface facilitates quantification and/or control of sample thickness by using only a predetermined spacer height in the closed configuration. For the non-planar inner surface of the plate, it is necessary to know exactly the topography of the inner surface, as well as the spacer height, so as to quantify and/or control the sample thickness in the closed configuration. Knowing the surface topology requires additional measurements and/or corrections, which is complicated, time consuming and costly.

  Plate surface flatness is generally the plate surface flatness variation for the final sample thickness (final thickness is the thickness in the closed configuration) relative to the final sample thickness. Is characterized by the term "relative surface flatness".

  In some embodiments, the relative surfaces are 0.01%, less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, Less than 20%, less than 30%, less than 50%, less than 70%, less than 80%, less than 100%, or a range between any two of these values.

  Plate Surface Parallelism In some embodiments, the two surfaces of the plate are significantly parallel to each other. In certain embodiments, the two surfaces of the plate are not parallel to each other.

  Plate Flex In some embodiments, the plate is flexible under compression of the CROF process. In some embodiments, under compression of the CROF process, the two plates are both flexible. In some embodiments, under compression of the CROF process, one plate is rigid and another is flexible. In some embodiments, both plates are rigid. In some embodiments, the two plates are both flexible, but different in flexibility.

  Plate Optical Clarity In some embodiments, the plate is optically transparent. In some embodiments, both plates are optically transparent. In some embodiments, one plate is optically transparent and another plate is non-transparent. In some embodiments, both plates are non-transparent. In some embodiments, the two plates are both optically transparent, but have different optical clarity. The optical clarity of a plate refers to some or the entire area of the plate.

  Surface Wettability In some embodiments, the plate has an inner surface that wets the sample, the transfer liquid, or both (ie, the contact angle is less than 90 degrees). In some embodiments, the two plates both have an inner surface that wets the sample, the transfer liquid, or both, and has the same or different wettability. In some embodiments, one plate has an inner surface that wets the sample, the transfer liquid, or both, and another plate has a non-wetting inner surface (ie, a contact angle of 90 degrees or greater). Wetting of the inner surface of the plate refers to some or the entire area of the plate.

  In some embodiments, the inner surface of the plate has other nano or microstructures for controlling the lateral flow of the sample during CROF. Nano- or microstructures include, but are not limited to, channels, pumps, etc. Nano and microstructures are also used to control the wetting properties of the inner surface.

2.3 Spacer Function of Spacer In the present invention, the spacer is configured to have one or any combination of the following functions and characteristics, and the spacer includes (1) the plate, the thickness of the sample or the sample. (2) The sample is compressed controlled open flow (CROF) to the plate surface to control the volume of interest (preferably the thickness control over the area of interest is accurate and/or uniform). (3) does not occupy a large surface area (volume) in a given sample area (volume), (4) reduces the sedimentation effect of particles or analytes in the sample? Or (5) to modify and/or control the wetting characteristics of the inner surface of the plate, (6) to identify the position of the plate, the size scale, and/or relevant information of the plate, or (7) It is configured to perform the above arbitrary combination.

  Spacer Architecture and Shape In order to achieve the desired sample thickness reduction and control, in certain embodiments, spacers are fixed on their corresponding plates. In general, the spacers may have any shape, and as long as the spacer can adjust the sample thickness in the CROF process, a certain shape has a certain function, eg, better uniformity. It is preferable for the realization of the characteristics, such as less overshoot at the time of pressing.

  The spacer is one spacer or a plurality of spacers. (Eg array). Some embodiments of multiple spacers are arrays of spacers (eg, columns), where the distance between the spacers is periodic or aperiodic, or in certain areas of the plate. It is periodic or aperiodic or has different distances in different areas of the plate.

  Spacers come in two types: open spacers and closed spacers. The open spacers are spacers that allow the sample to flow through the spacers (ie, the sample flows around and passes around the spacers, eg, posts as spacers) and the closed spacers stop the sample flow. A spacer (ie, the sample cannot flow out of the spacer, eg, an annular spacer and the sample is in the annulus). Both types of spacers use their height to adjust the final sample thickness in the closed configuration.

  In some embodiments, the spacers are open spacers only. In some embodiments, the spacers are closed spacers only. In some embodiments, the spacer is a combination of open and closed spacers.

  The term "columnar spacer" means that the spacer exhibits a columnar shape, which refers to an object having a height and a lateral shape, the height and the lateral shape of which surrounds it during compression open flow. Through which the sample can flow.

  In some embodiments, the lateral shape of the columnar spacers is (i) circular, oval, rectangular, triangular, polygonal, ring-shaped, star-shaped, letter-shaped (eg, L-shaped, C-shaped, letter A-A). Z), a numeral shape (for example, a shape such as 0, 1, 2, 3, 4,... 9), (ii) a shape in the group (i) having at least one rounded corner, (iii) a zigzag shape or a free space. The shape in the group (i) with edges and the shape in the group (iv) (i) and (ii) and (iii) any superposition. For multiple spacers, the different spacers may have different lateral shapes and sizes and different distances between adjacent spacers.

  In some embodiments, the spacers may be and/or include posts, cylinders, beads, balls, and/or other suitable geometric shapes. The lateral shape and dimensions of the spacers (ie, across their respective plate surfaces), in some embodiments, (i) the geometry of the spacers causes obvious errors when measuring sample thickness and volume. And (ii) the spacer geometry has the limitation that it does not stop the flow of sample between the plates (ie, it is not in a closed form). However, in some embodiments, some spacers, which are hermetically sealed spacers, are needed to limit sample flow.

  In some embodiments, the spacer shape has rounded corners. For example, one rectangular spacer has one, some, or all rounded corners, or all corners are rounded corners (similar to a circle but not 90 degree corners). In general, rounded corners may facilitate spacer manufacture and may reduce damage to biological material.

  The sidewalls of the pillars may be straight, curved, sloping, or have different shapes depending on the portions of the sidewalls. In some embodiments, the spacers are pillars having various lateral shapes, sidewalls and pillar height to pillar side area ratios.

  In one preferred embodiment, the spacer has a post that allows open flow.

  Spacer Material In the present invention, the spacer is typically made of any material that can be used with two plates to adjust the thickness of a sample of related volume. In some embodiments, the spacer material and the plate material are different. In some embodiments, the material used for the spacer is at least the same as some of the material used for the at least one plate.

  The spacer is made of a single material, a composite material, multiple materials, multiple layers of material, alloys, or combinations thereof. The material used for the plate is an inorganic material, an organic material, or a combination thereof, examples of which are described in paragraphs Mat-1 and Mat-2. In one preferred embodiment, the spacers are made of the same material as the plates used in CROF.

  Mechanical Strength and Flexibility of Spacers In some embodiments, the spacers are sufficiently mechanically strong that during the plate compressing and closing configuration process, the spacer height of the plate when in the open configuration is increased. It is the same as or substantially the same as the height. In some embodiments, the difference between the spacers in the open and closed configurations may be characterized or predetermined.

  The material used for the spacer may be rigid, flexible, or any flexible in between. Rigidity is related to the pressing force that places the plate in a closed configuration, at which the spacer is considered rigid when the spatial height is deformed by less than 1%, and otherwise flexible. Conceivable. If the spacer is made of a flexible material, the final sample thickness in the closed configuration can still be predetermined based on the pressing force and the mechanical strength of the spacer.

  Spacers within the Sample In order to achieve the desired reduction and control over the sample thickness, and in particular, to achieve excellent sample thickness uniformity, in certain embodiments, the spacers are within the sample or of the relevant volume. It is placed in the sample. In some embodiments, there are one or more spacers within the sample or related volume of sample, with an appropriate distance between the spacers. In certain embodiments, at least one of the spacers is located in the sample and at least two of the spacers are located in the sample or in a relevant volume of the sample, or at least "n" The spacers are located in the sample or in the relevant volume of the sample, where "n" is determined by sample thickness uniformity between CROFs or required sample flow characteristics.

  Spacer Height In some embodiments, all spacers have the same predetermined height. In some embodiments, the spacers have different predetermined heights. In some embodiments, the spacers are divided into groups or regions, with each group or region having a respective spacer height. Also, in certain embodiments, the spacer is such that the predetermined height is the average height of the spacer. In some embodiments, the spacers have substantially the same height. In some embodiments, the percentage of spacers have the same height.

  The height of the spacers is selected according to the desired adjusted final sample thickness and residual sample thickness. The spacer height (predetermined spacer height) and/or sample thickness is 3 nm or less, 10 nm or less, 50 nm or less, 100 nm or less, 200 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1 μm or less, 2 μm or less. 3 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 50 μm or less, 100 μm or less, 150 μm or less, 200 μm or less, 300 μm or less, 500 μm or less, 800 μm or less, 1 mm or less, 2 mm or less, 4 mm or less, or these values Is a range between any two.

  The spacer height and/or sample thickness is in one preferred embodiment between 1 nm and 100 nm, in another preferred embodiment between 100 nm and 500 nm, one single preferred embodiment. , Between 500 nm and 1000 nm, in another preferred embodiment between 1 μm (ie 1000 nm) and 2 μm, in another preferred embodiment between 2 μm and 3 μm, another preferred embodiment , Between 3 μm and 5 μm, in another preferred embodiment, between 5 μm and 10 μm, in another preferred embodiment, between 10 μm and 50 μm, in one single preferred embodiment, 50 μm ˜100 μm.

  In some embodiments, the spacer height and/or sample thickness is (i) equal to or slightly greater than the minimum dimension of the analyte, or (ii) equal to the maximum dimension of the analyte, or Slightly larger than that. "Slightly greater" means about 1% to 5% greater and any number between two values.

  In some embodiments, the spacer height and/or sample thickness is greater than the minimum dimension of the analyte (eg, an analyte having an anisotropy shape) but less than the maximum dimension of the analyte.

  For example, red blood cells have a disc shape with a minimum dimension of 2 μm (disc thickness) and a maximum dimension of 11 μm (disc diameter). In one embodiment of the invention, the spacer provides an inner surface spacing of the plates within the relevant area of 2 μm (equal to the smallest dimension) in one embodiment and 2.2 μm in another embodiment. Or in another embodiment 3 μm (50% larger than the smallest dimension) but smaller than the largest dimension of the red blood cells. Such an embodiment has certain advantages in counting red blood cells. In one embodiment, for counting red blood cells, the undiluted whole blood sample is averaged within the interval by setting the inner surface interval to 2 μm or 3 μm to any value between the two values. By way of limitation, each red blood cell (RBC) does not overlap with other red blood cells, thereby allowing accurate red blood cell counting. (A lot of overlap between RBCs can lead to serious counting errors).

  In some embodiments, the present invention uses plates and spacers to control not only sample thickness, but also the orientation and/or surface density of the analyte/entity in the sample when the plate is in the closed configuration. .. The thin thickness of the sample results in less analyte/entity per surface area (ie, lower surface concentration) when the plate is in the closed configuration.

  Spacer Lateral Dimension In the case of an open spacer, the lateral dimension can be characterized by its lateral dimension in two orthogonal directions, x and y (sometimes called width). The lateral dimensions of the spacers in each direction may be the same or different. In some embodiments, the lateral dimension in each direction (x or y) is...

  In some embodiments, the ratio of the lateral dimensions in the x and y directions is 1, 1.5, 2, 5, 10, 100, 500, 1000, 10,000, or any of these values. It is the range between the two. In some embodiments, different ratios are used to adjust the flow direction of the sample, the higher the ratio the more the flow is along one direction (larger size direction).

  In some embodiments, the different lateral dimensions of the spacer in the x and y directions allow (a) the spacer to be used as a scale marker to indicate the orientation of the plate, and (b) the spacer to be used to provide a favorable flow Used to make more samples in a direction, or both.

  In one preferred embodiment, period, width, and height.

  In some embodiments, all spacers have the same shape and size. In some embodiments, each spacer has a different lateral dimension.

  In the case of a closed spacer, in some embodiments, the lateral shape and size of the interior is selected according to the total volume of the sample closed by the closed spacer, of which volume and size have already been described in this disclosure. In certain embodiments, the outer lateral shape and size are selected depending on the strength required to support the pressure of the liquid against the spacer and the compression pressure that presses the plate.

Aspect Ratio of Height to Average Lateral Dimension of Columnar Spacers In certain embodiments, the aspect ratio of height to average lateral dimension of the columnar spacers is 100,000, 10,000, 1,000, 100, 10, It is 1, 0.1, 0.01, 0.001, 0.0001, 0.00001, or a range between any two of these values.

  Spacer Height Accuracy Spacer height accuracy should be precisely controlled. The relative accuracy of the spacers (ie, the ratio of the deviation to the desired spacer height) is 0.001% or less, 0.01% or less, 0.1% or less, 0.5% or less, 1% or less, 2 % Or less, 5% or less, 8% or less, 10% or less, 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less. , 99.9% or less, or a range between any of these numbers.

  Distance Between Spacers The spacer may be one spacer or multiple spacers on the plate or in the sample of the relevant area. In some embodiments, the spacers on the plate are arranged and/or arranged in an array, the array being a periodic, aperiodic array, or periodic at some positions of the plate. , Aperiodic at other locations.

  In some embodiments, the periodic array of spacers comprises a grid of squares, rectangles, triangles, hexagons, polygons, or any combination thereof, where the combinations are different spacer grids at different positions of the plate. Means to have.

  In some embodiments, the distance between the spacers in the spacer array is periodic in at least one direction of the array (ie, the distance between the spacers is uniform). In some embodiments, the distance between the spacers is configured to improve uniformity between plate spacing in the closed configuration.

  The distance between adjacent spacers (that is, the distance between spacers) is 1 μm or less, 5 μm or less, 10 μm or less, 20 μm or less, 30 μm or less, 40 μm or less, 50 μm or less, 60 μm or less, 70 μm or less, 80 μm or less, 90 μm or less, 100 μm or less. , 200 μm or less, 300 μm or less, 400 μm or less, or a range between any two of these values.

  In certain embodiments, the distance between the spacers is 400 or less, 500 or less, 1 mm or less, 2 mm or less, 3 mm or less, 5 mm or less, 7 mm or less, 10 mm or less, or any range between these values. .. In certain embodiments, the distance between the spacers is 10 mm or less, 20 mm or less, 30 mm or less, 50 mm or less, 70 mm or less, 100 mm or less, or any range between these values.

  The distance between adjacent spacers (ie, the distance between spacers) is such that in a closed configuration of the plate, for a given characteristic of the plate and the sample, there is some variation in sample thickness between two adjacent spacers. In the form up to 0.5%, 1%, 5%, 10%, 20%, 30%, 50%, 80%, or any range between these values, or in certain embodiments, Selected to be up to 80%, 100%, 200%, 400%, or a range between any two of these values.

  Obviously, in order to maintain a given sample thickness variation between two adjacent spacers, closer spacer distances are needed when more flexible plates are used.

  Specify the accuracy of the distance between the spacers.

  In one preferred embodiment, the spacers are periodic square arrays, of which the spacers have a height of 2 μm to 4 μm, an average lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 1 μm to 100 μm. It has a columnar shape.

  In one preferred embodiment, the spacers are periodic square arrays, the spacers having a height of 2 μm to 4 μm, a mean lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 100 μm to 250 μm. belongs to.

  In one preferred embodiment, the spacers are periodic square arrays, the spacers having a height of 4 μm to 50 μm, a mean lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 1 μm to 100 μm. belongs to.

  In one preferred embodiment, the spacers are periodic square arrays, of which the spacers have a height of 4 μm to 50 μm, an average lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 100 μm to 250 μm. belongs to.

  The period of the spacer array is in one preferred embodiment between 1 nm and 100 nm, in another preferred embodiment between 100 nm and 500 nm, and in one single preferred embodiment between 500 nm and 1000 nm. And in another preferred embodiment between 1 μm (ie 1000 nm) and 2 μm, in another preferred embodiment between 2 μm and 3 μm, in another preferred embodiment between 3 μm and 5 μm. In another preferred embodiment between 5 μm and 10 μm, in another preferred embodiment between 10 μm and 50 μm, in one single preferred embodiment between 50 μm and 100 μm, In one single preferred embodiment, between 100 μm and 175 μm, and in one single preferred embodiment, between 175 μm and 300 μm.

The spacer density spacer is greater than 1 / [mu] m ^2, greater than 1/10 [mu] m ^2, greater than 1/100 [mu] m ^2, greater than 1/500 [mu] m ^2, greater than 1/1000 .mu.m ^2, 1/5000 .mu.m greater than ^2, greater than one /0.01Mm ^2, greater than one 0.1 mM ^2, greater than one / 1 mm ^2, greater than 1/5 mm ^2, 1 greater than pieces / 10 mm ^2, greater than 1/100 mm ^2, greater than 1/1000 mm ^2, is one / or greater than 10000 mm ^2, or any two ranges between these values The surface density is arranged on each plate.

  (3) The spacer is configured not to occupy an apparent surface area (volume) within a predetermined sample area (volume).

  Ratio of Spacer Volume to Sample Volume In some embodiments, to achieve certain advantages, the ratio of spacer volume (ie, spacer volume) to sample volume (ie, sample volume) and/or sample association The ratio of the volume of the spacer in the sample of the relevant volume to the volume of is controlled. The advantages include, but are not limited to, sample thickness control uniformity, analyte uniformity, sample flow characteristics (ie, flow rate, flow direction, etc.).

  In certain embodiments, the ratio of the spacer volume to the sample volume and/or the volume of the spacer inside the sample of the relevant volume to the relevant volume of the sample is less than 100%, at most 99%. , At most 70%, at most 50%, at most 30%, at most 10%, at most 5%, at most 3%, at most 1%, at most 0.1% , At most 0.01%, at most 0.001%, or any range between these values.

  Spacers Fixed to Plates In the present invention, the distance and orientation between the spacers that play an important role are preferably maintained in the process of moving the spacers from the open configuration to the closed configuration and/or preferably in the open configuration. To a closed configuration prior to the process.

  In some embodiments of the invention, the spacers are fixed on one plate prior to placing the plate in the closed configuration. The term "spacer is fixed to its corresponding plate" means that the spacer is adhered onto the plate and the adhesion is maintained during the use of the plate. One example of "a spacer is fixed to its respective plate" is that the spacer is made monolithically from a single plate material, and the position of the spacer with respect to the plate surface does not change. An example of "the spacers are not secured to their respective plates" is that the spacers are adhered to the plate by an adhesive, but the adhesive does not keep the spacers in their original position on the plate surface during use of the plate (ie, , The spacer moves away from its original position on the plate surface).

  In some embodiments, at least one spacer is fixed to its corresponding plate. In certain embodiments, at least two spacers are fixed to their corresponding plates. In certain embodiments, most spacers are fixed to their corresponding plates. In a particular embodiment, all spacers are fixed to their corresponding plates.

  In some embodiments, the spacer is integrally fixed to the plate.

  In some embodiments, the spacers are secured to their corresponding plates by one or any of the methods and/or configurations of gluing, laminating, fusing, imprinting, etching.

  The term "imprint" means to immobilize (ie, emboss) a material to secure the spacer and plate together to form the spacer on the plate surface. The material may be a single layer material or a multilayer material.

  The term "etching" means fixing the spacer and the plate together by etching one material to form the spacer on the plate surface. The material may be a single layer material or a multilayer material.

  The term “fusion” means that the spacer and the plate are integrally fixed by adhering the spacer and the plate together, and the raw materials of the spacer and the plate are fused with each other, and the two materials after the fusion are performed. It means that there is a clear material boundary between.

  The term “bonding” means integrally fixing the spacer and the plate by integrally bonding the spacer and the plate with an adhesive.

  The term "adhesion" means connecting the spacer and the plate together.

  In some embodiments, the spacer and plate are made of the same material. In another embodiment, the spacers and plates are made of different materials. In another embodiment, the spacer and plate are integrally formed. In another embodiment, one end of the spacer is fixed on its corresponding plate while the ends are open to accommodate different configurations of the two plates.

  In another embodiment, each spacer is independently connected to the corresponding plate by at least one of gluing, bonding, fusing, imprinting, etching. The term "independent" means that one spacer is adhered to, bonded to, fused to, imprinted with, and etched to the corresponding plate in the same or different manner selected from the corresponding way. It means to fix it to the plate.

  In some embodiments, at least one distance between two spacers is predetermined ("distance between predetermined spacers" means that the distance is known when the user uses the plate. means).

  In some embodiments of all methods and apparatus described herein, there are additional spacers in addition to the fixed spacers.

  Specific Sample Thickness In the present invention, a large plate holding force (ie, two plates are brought together by small plate spacing (for a given sample area) or large sample area (for a given plate spacing), or both. It was observed that the holding power) can be obtained.

  In some embodiments, at least one plate is transparent in the area surrounding the area of interest and each plate contacts the sample in a closed configuration; in the closed configuration the inner surface of the plate is a spacer. Are substantially parallel to each other, except that the positions having? Are substantially parallel to each other; the inner surface of the plate is configured to be substantially planar; or any combination thereof.

2.4 Final Sample Thickness and Uniformity In some embodiments, being significantly flat is determined with respect to the final sample thickness, and depending on the embodiment and application, the ratio to sample thickness is: Less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, or less than 10%, or a range between any two of these values. .

  In some embodiments, flatness to sample thickness is less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, 20. It may be less than %, less than 50%, or less than 100%, or a range between any two of these values.

  In some embodiments, being significantly flat means that the surface flatness variation itself (as measured from the average thickness) is less than 0.1%, less than 0.5%, less than 1%, 2 %, less than 5%, or less than 10%, or a range between any two of these values. Generally, the flatness with respect to the sample thickness is less than 0.1%, less than 0.5%, less than 1%, less than 2%, less than 5%, less than 10%, less than 20%. It may be small, less than 50%, or less than 100%, or a range between any two of these values.

2.5 Method of Manufacturing Spacers Spacers can be manufactured into plates in a variety of ways using lithographic, etching, embossing (nanoimprint), deposition, lift-off, fusion, or combinations thereof. In some embodiments, the spacers are embossed or imprinted directly on the plate. In some embodiments, spacers are imprinted in the material (eg, plastic) deposited on the plate. In a particular embodiment, the spacers are manufactured by directly imprinting the surface of the CROF plate. Nanoimprinting can be done by roll-to-roll technology using a low-line printer or by winding into planar nanoimprints. Such a process has great economic advantages and thus lower costs.

  In some embodiments, spacers are deposited on the plate. The deposition may be evaporation, sticking, or lift-off. When pasting, first the spacers are manufactured on the carrier and then the spacers are transferred from the carrier to the plate. When lifted off, the plate is first deposited with removable material and holes are formed in the material such that the bottom of the hole exposes the plate surface, then spacer material is deposited in the hole and then removable. Material is removed, leaving only spacers on the plate surface. In some embodiments, the spacers deposited on the plate are fused with the plate. In some embodiments, the spacers and plates are manufactured in a single process. The single process includes imprinting (ie, embossing, molding) or synthesis.

  In some embodiments, at least two spacers are secured to their corresponding plates in different ways, optionally the different methods include at least one of deposition, laminating, fusing, imprinting, and etching. ..

  In some embodiments, one or more spacers are affixed to corresponding plates by a manufacturing method such as laminating, fusing, imprinting, or etching or any combination thereof.

  In some embodiments, manufacturing methods for forming such integral spacers in plates include laminating, fusing, imprinting, etching, or any combination thereof.

2.6 Scale Markers The term “scale markers” refers to scale markers that can assist in the quantification (ie, dimensional measurement) or control of the relative volume of a relevant area and/or sample. In some embodiments, the scale marker is located on the first plate or the second plate, two plates, one surface of the plate, two surfaces of the plate, between the plates, near the plate, or Any combination of them. In some embodiments, the scale markers are on the first plate or the second plate, on both plates, on one surface of the plate, on both surfaces of the plate, between the plates, between the plates. It may be fixed nearby or any combination thereof. In some embodiments, scale markers are deposited on the first plate or the second plate, two plates, one surface of the plate, two surfaces of the plate, between the plates, in the vicinity of the plate, or Or any combination thereof. In some embodiments, some spacers are fixed and some spacers are deposited.

  In some embodiments, the scale markers are etched scale markers, deposited material, or printed material. In certain embodiments, the material is a material that has light absorption, light reflection, light emission, or any combination thereof.

  In some embodiments, the scale markers are one or more objects that have known dimensions and/or known separation distances. Examples of subjects include, but are not limited to, rectangular, cylindrical, or circular.

  In some embodiments, the scale markers have dimensions within the nanometer (nm), micrometer (μm) or millimeter (mm), or other size range.

  In some embodiments, the scale marker is a ruler, which has a scale marker configured to measure the dimension of the subject. In some embodiments, the scale markers are in the nanometer (nm), micrometer (μm) or millimeter (mm), or other size range. In some embodiments, the scale markers are etched scale markers, deposited material or printed material. In some embodiments, the material of the scale marker is light absorbing, light reflecting, light scattering, light interfering, light diffracting, light emitting, or any combination thereof.

  In some embodiments, the marker is a spacer, which has the dual function of "adjusting sample thickness" and "providing scale marking and/or dimensional scaling." For example, rectangular spacers with known dimensions or two spacers with known spacing can be used to measure the dimensions with respect to the sample around the spacers. The measured sample size allows the volume of the sample of the relevant volume to be calculated.

  In some embodiments, the scale markers are configured to partially define the boundaries of the relevant volume of the sample.

  In some embodiments, the at least one scale marker is configured to have a known dimension that is parallel to the plane of the lateral region of the sample of relevant volume.

  In some embodiments, the at least one pair of scale markers are separated by a known distance that is parallel to the plane of the lateral region.

  In some embodiments, scale markers are configured for use in optical detection.

  In some embodiments, each scale marker independently has at least one function of light absorption, light reflection, light scattering, light diffraction and light emission.

  In some embodiments, the scale markers are arranged in a regular array with known lateral spacing.

  In some embodiments, each scale marker independently has a lateral contour of at least one of square, rectangle, polygon, and circle.

  In some embodiments, at least one scale marker is glued, laminated, fused, imprinted and etched to one plate.

  In some embodiments, at least one scale marker is one spacer.

  In some embodiments, some spacers also serve as scale markers to quantify the relevant volume of the sample.

  In certain embodiments, binding sites (for immobilizing analytes), storage sites, etc. are used as scale markers. In one embodiment, the site having a known lateral dimension interacts with light that produces a detectable signal that reproduces the known lateral dimension, thereby providing a scale marker.

  In another embodiment, the dimension of the site is predetermined prior to the CROF process, and when the plate is in the closed configuration, the thickness of the sample portion located at the site is more pronounced than the average lateral dimension of the site. By controlling the incubation time to be small, and then after incubation (1), most of the analyte/entity bound to the binding site will come from the sample volume atop the binding site, or (2) The reagent mixed (diffused) in the sample volume on top of most of the binding sites comes from the storage site. In these cases, the relevant volume of the sample for binding or mixing reagents is approximately equal to the product of the area of a given site and the thickness of the sample at that site. The main reason for this is that for a given incubation time there is not enough time for the analyte/entity in the sample volume outside the relevant volume to diffuse to the binding site or the reagent on the storage site is relevant. There is not enough time to diffuse into the sample volume outside some volume.

  In one example of a method of measuring and/or controlling the area and volume of interest by using sites with known dimensions and by limiting the incubation time, the assay involves the first plate of the CROF process (that The surface is larger than the binding site) and has a binding site of 1000 μm×1000 μm (ie the area containing the capture agent), in the closed configuration of the plate the sample containing the analyte is located above the binding site, The sample has a thickness of about 20 μm (in the region of the binding site) and an area greater than the binding site, and is incubated for a time equal to the diffusion time of the total sample thickness of the target analyte/entity. In this case, most of the analyte/entity bound to the binding site came from the sample volume at the top of the binding site, 1000 μm×1000 μm×20 μm=0.02p, because 20 μm away from the binding site. There is no time (statistically) to diffuse to the binding site in the sample part. In this case, the signal, when measured after incubation due to the analyte/entity captured by the binding site, is of interest based on the relevant area and relevant volume information (provided by the binding site). The concentration of the analyte/entity in a region and a relevant volume of the sample can be determined. Analyte concentration is quantified by dividing the amount of analyte captured by the binding site by the relevant volume.

  In some embodiments, the volume of interest is approximately equal to the product of the binding site area and the sample thickness, and the concentration of the target analyte in the sample is related to the amount of analyte captured by the binding site. It is almost equal to the result divided by the sample volume. The greater the ratio of the binding site size to the sample thickness, the more accurate the method of quantifying the target analyte volume (incubation time is when the target analyte in the sample diffuses to a distance of about sample thickness). Suppose it is the required time).

  Spreading Time in CROF In the present invention, in all the methods and devices of spreading the sample by two plates, the time required to spread the sample to the final thickness in the closed configuration is less than 0.001 seconds, 0.01 Seconds, 0.1 seconds, 1 second, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, 100 seconds, 150 seconds, 200 seconds, 300 seconds, 500 seconds, 1000 seconds, or values thereof Is a range between any two.

  In the method and apparatus of all paragraphs for spreading the sample with two plates, in one preferred embodiment, the time required to spread the sample to the final thickness in the closed configuration is less than 0.001 seconds, 0.01 seconds. , 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, 100 seconds, 150 seconds, or a range between any two of these values. ..

  In the method and apparatus of all paragraphs for spreading the sample with two plates, in one preferred embodiment, the time required to spread the sample to the final thickness in the closed configuration is less than 0.001 seconds, 0.01 seconds. , 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, 60 seconds, 90 seconds, or a range between any two of these values.

  In the method and apparatus of all paragraphs for spreading the sample with two plates, in one preferred embodiment, the time required to spread the sample to the final thickness in the closed configuration is less than 0.001 seconds, 0.01 seconds. , 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, 20 seconds, 30 seconds, or a range between any two of these values.

  In the method and apparatus of all paragraphs for spreading the sample with two plates, in one preferred embodiment, the time required to spread the sample to the final thickness in the closed configuration is less than 0.001 seconds, 0.01 seconds. , 0.1 seconds, 1 second, 3 seconds, 5 seconds, 10 seconds, or a range between any two of these values.

  In the method and apparatus of all paragraphs for spreading the sample with two plates, in one preferred embodiment, the time required to spread the sample to the final thickness in the closed configuration is less than 0.001 seconds, 0.01 seconds. , 0.1 seconds, 1 second, 3 seconds, or any two of these values.

  The embodiments described in Section 3 and any combination thereof apply to (ie are combined with) other embodiments throughout the description of the invention.

  In one preferred embodiment, the spacers are made integrally with the X-plate and made of the same material by coining a thin plastic film (eg, nanoimprint) using a mold.

  In one preferred embodiment, the spacer is made integrally with the X-plate by coining a thin plastic film (eg, nanoimprint) using a mold and made of the same material, The thickness is 50 μm to 500 μm.

  In one preferred embodiment, the spacer is made integrally with the X-plate by coining a thin plastic film (eg, nanoimprint) using a mold and made of the same material, The thickness is 50 μm to 250 μm.

  In one preferred embodiment, the spacers are made integrally with the X-plate and made of the same material, the thickness of the X-plate being between 50 μm and 500 μm.

  In one preferred embodiment, the spacer is made integrally with the X-plate by using a mold and a thin plastic film and made of the same material, the thickness of the X-plate is between 50 μm and 250 μm. is there.

  In one preferred embodiment, the spacer is made integrally with the X-plate by coining a thin plastic film with a mold (eg nanoimprinting) and made of the same material, the plastic film being , PMMA (polymethylmethacrylate) or PS (polystyrene).

  In one preferred embodiment, the spacer is made integrally with the X-plate by coining a thin plastic film with a mold (eg nanoimprinting) and made of the same material, the plastic film being , PMMA (polymethylmethacrylate) or PS (polystyrene), and the thickness of the X-plate is 50 μm to 500 μm.

  In one preferred embodiment, the spacer is made integrally with the X-plate by coining a thin plastic film with a mold (eg nanoimprinting) and made of the same material, the plastic film being , PMMA (polymethylmethacrylate) or PS (polystyrene), and the thickness of the X-plate is 50 μm to 250 μm.

  In one preferred embodiment, the spacer is made integrally with the X-plate by coining a thin plastic film with a mold (eg nanoimprinting) and made of the same material, the plastic film being , PMMA (polymethylmethacrylate) or PS (polystyrene), the spacers have a square or rectangular shape and have the same spacer height.

  In one preferred embodiment, the spacer has a square or rectangular shape (with or without circular corners).

  In one preferred embodiment, the spacers have square or rectangular pillars, the pillar width (width of the spacer in each lateral direction) is between 1 μm and 200 μm, and the pillar period (ie, spacer period) is 2 μm. ˜2000 μm and the height of the pillars (ie the height of the spacers) is between 1 μm and 100 μm.

  In one preferred embodiment, the spacers made of PMMA or PS have square or rectangular pillars, the width of the pillars (width of the spacer in each lateral direction) is between 1 μm and 200 μm, and the period of the pillars ( That is, the spacer period) is 2 μm to 2000 μm, and the height of the columns (that is, the spacer height) is 1 μm to 100 μm.

  In a preferred embodiment, the spacer is manufactured integrally with the X-plate and is made of a plastic material, said spacer having square or rectangular columns, the width of the columns (width of the spacer in each lateral direction). ) Is from 1 μm to 200 μm, the column period (ie, spacer period) is from 2 μm to 2000 μm, and the column height (ie, spacer height) is from 1 μm to 100 μm.

  In one preferred embodiment, the spacer is manufactured integrally with the X-plate and made of the same material, with the spacer having square or rectangular columns, the width of the columns (width of the spacer in each lateral direction). ) Is between 1 μm and 200 μm, the period of the columns (ie the spacer period) is between 2 μm and 2000 μm, and the height of the columns (ie the height of the spacers) is between 1 μm and 10 μm.

  In one preferred embodiment, the spacers are made integrally with the X-plate and made of the same material selected from PS or PMMA or other plastics, said spacers having square or rectangular posts, Has a width (width of the spacer in each lateral direction) of 1 μm to 200 μm, a period of columns (ie, spacer period) is 2 μm to 2000 μm, and a height of columns (ie, height of spacers) is 10 μm to 50 μm.

  In one preferred embodiment of the CROF device, one plate is an X plate, the other plate is a planar thin film, the thickness of at least one plate is between 10 μm and 250 μm, and the spacer is on the X plate. Fixed, the plate and spacer materials may be the same or different and are made of PMMA (polymethylmethacrylate), PS (polystyrene) or similar material with mechanical properties similar to PMMA or PS ..

  In one preferred embodiment of the CROF device, one plate is an X plate, the other plate is a planar thin film, the thickness of at least one plate is between 250 μm and 500 μm, and the spacer is on the X plate. Fixed, the plate and spacer materials may be the same or different and are made of PMMA (polymethylmethacrylate), PS (polystyrene) or similar material with mechanical properties similar to PMMA or PS .

  In one preferred embodiment of the CROF device, one plate is an X plate, the other plate is a planar thin film, the thickness of at least one plate is between 10 μm and 250 μm, and the spacer is on the X plate. Fixed, the spacer is an array of square or rectangular columns, the column width (width of the spacer in each lateral direction) is between 1 μm and 200 μm, the column period (ie the spacer period) is between 2 μm and 2000 μm, The height of the pillar (that is, the height of the spacer) is 1 μm to 100 μm, the material of the plate and the spacer may be the same or different, and PMMA (polymethylmethacrylate), PS (polystyrene) or PMMA. Alternatively, it is made of a similar material with mechanical properties similar to PS.

  “Similar” in the above paragraphs means that the difference in mechanical properties is within 60%.

  Guard Ring In some embodiments, a guard ring is provided to prevent the sample from escaping the plate surface. In some embodiments of the guard ring, it is a wall sealed about the sample area. The wall height is or is not equal to the spacer height. The wall may be separated from the sample measurement area.

  The movable plate in the CROF process may include and/or be coupled to a hinge, stage, or some other positioning system, which system converts the plate between an open configuration and a closed configuration. Is composed of. The movable plate leaves an opening for entry into the space between the plates, provided that the at least one joint and/or the at least one plate are sufficiently flexible to achieve the open and closed configurations ( For example, a sample can be inserted and/or removed) and associated with one or more fittings. Membrane pumps are not considered moving plates.

3. Uniform plate spacing and sample thickness (U)
In many applications of the CROF process, it is desirable to improve the thickness of the sample in the closed configuration by improving the uniformity of the plate spacing, especially when the spacing is on the micrometer and/or nanometer scale. Good homogeneity can improve assay homogeneity. The present invention provides a method for improving uniformity.

  Factors that reduce the uniformity of plate spacing in CROF include (a) local curvature of the plate, (b) unevenness of the inner surface of the plate, and (c) dust. The smaller the spacing of the final plates, the more severe the effect of these factors.

  In order to improve the pitch uniformity (and therefore the sample thickness), the present invention provides a specific design (mechanical strength, thickness, etc.) in the plate, such as spacer size, number of spacers, spacer layout, spacers. Use the accuracy of the distance between them, the height of the spacers, etc. to eliminate the factors that cause non-uniformity.

Inner Surface Smoothness 3.1 Use of Distance Between Spacers to Achieve Uniform Sample Thickness of Flexible Plates In some applications making one or two of the CROF plates flexible Is desirable. However, as shown in FIG. 5a, for a flexible plate (eg, a plastic film), if the distance between the spacers is too large, the flexibility of the plate in the CROF process will be in the position between two adjacent spacers. Local curvature (e.g., indentation, i.e., inward curvature), resulting in poor sample thickness uniformity. Low sample thickness uniformity has many drawbacks, such as large errors in determining sample volume and/or analyte concentration, variations in incubation time, and the like.

  One embodiment of the present invention provides a solution that reduces local curvature by using suitable spacer-to-spacer distances, thus reducing variations in final sample thickness. As shown in FIG. 5, if the distance between the spacers is too large, the CROF device will have one rigid plate with a flat sample surface and one flexible plate with a local curvature between two adjacent spacers. (FIG. 5a). To reduce local curvature, the distance between the spacers is set below the critical curvature span of the flexible plate (Fig. 5b). If both plates are flexible, the distance between the spacers should be less than the minimum critical span of the two plates.

U1. A method for uniformly adjusting the thickness of a sample of related volume using two plates, comprising: (a) obtaining a sample, wherein the thickness of the sample of related volume is Adjusted, step,
(B) Obtaining two plates that are movable relative to each other in different configurations, one or two plates being flexible and one or two of said plates being spacers. The spacers have a predetermined distance and height between the spacers, and each spacer is fixed to its corresponding plate,
(C) depositing a sample on one or two of the previous plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacers, is a step,
(D) after (c) the step of spreading the sample by placing the plates in a closed configuration, wherein the plates face each other in the closed configuration and the spacer and the associated volume of sample are located between the plates. And the thickness of the sample of the relevant volume is adjusted by the plates and spacers,
For a given plate, the spacer is configured to have a variation of the thickness of the relevant volume of the sample for a given region of less than a predetermined value, the relevant volume being a fraction of the volume of the sample or the entire volume. Is the way.

  In the method of paragraph U1, spacer and plate configuration includes selecting an appropriate spacer distance. In some embodiments, by selecting the distance between the spacers, for allowed sample thickness variations, two plates given, and for the compression method, the curvature of the two plates due to the compression method is allowed. It is less than the fluctuation of the sample thickness. When in the closed configuration, the adjusted sample thickness can be less than the maximum thickness of the sample when the plate is in the open configuration.

U2. A device for adjusting the thickness of a sample of related volume, comprising:
A first plate and a second plate moveable with respect to each other in different configurations,
One or two plates are flexible, and one or two of said plates comprises spacers, said spacers having a predetermined distance and height between spacers, each spacer being attached to its corresponding plate. Fixed,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration, which is constructed after the sample is deposited in an open configuration, in which the plates face each other and the spacer and associated volume of sample are located between the plates and are associated. The thickness of the volume sample is controlled by plates and spacers,
For a given plate, the spacer is configured to have a thickness variation for one region of the thickness of the relevant volume of the sample that is less than a predetermined value, the relevant volume being a fraction of the volume of the sample. Or a device that is the entire volume.

  In the apparatus of paragraph U2, spacer and plate configuration includes selecting an appropriate spacer distance. In some embodiments, by selecting the distance between the spacers, for allowed sample thickness variations, two plates given, and for the compression method, the curvature of the two plates due to the compression method is allowed. It is less than the fluctuation of the sample thickness. When in the closed configuration, the adjusted sample thickness can be less than the maximum thickness of the sample when the plate is in the open configuration.

  In some embodiments, the distance between the small spacers is further reduced by the distance between the spacers being less than the curvature of the plate between the two spacers, such as a flexible thin film (eg, a plastic sheet with a thickness of 100 μm). ) Can be used.

  In some embodiments, between the spacers for the critical bending span of the plate, for the maximum bending allowed for a given flexible plate to provide uniform sample thickness over large areas in the closed configuration. The ratio of distances is 0.001% maximum, 0.001% maximum, 0.001% maximum, 0.01% maximum, 0.1% maximum, 1% maximum, 10% maximum, 20% maximum, 50% maximum. , 70% maximum, 100% maximum, or a range between any two of these values.

3.2 Use of Flexible Plates and Spacers to Overcome the Impact of Dust in CROF The challenge that needs to be overcome in the CROF process is that dust whose thickness is greater than the height of the spacer is the desired final plate of the spacer. It is possible to destroy the regulatory action to achieve the spacing (and therefore the final thickness of the sample) (shown in Figure 6a). When using two rigid plates, this dust can destroy the spacer adjustment over the plate area.

  Certain embodiments of the present invention use an appropriate distance between the flexible plate and the spacer to limit the effect of dust in a small area around the dust while at the same time the area outside the small area is the spacer. The problem is solved by allowing to have a set (adjusted) final plate spacing and sample thickness.

  To overcome the effects of dust, one flexible plate with suitable flexibility was used to limit the area of the dust and to which it was fixed, as shown in FIG. 6b. For use with rigid plates. FIG. 6c shows another embodiment for reducing the effect of dust, in which the spacers are fixed on the flexible plate. Obviously, in other embodiments, the two plates are both flexible.

  From the thickness and mechanical properties of the plate, the appropriate plate flexibility that minimizes the effects of dust in the CROF process can be selected. Based on the examination shown in the example, the preferred embodiment is as follows.

U3. A method for minimizing the effect of dust on adjusting the thickness of a sample of relevant volume, comprising:
(A) obtaining a sample, wherein the thickness of the relevant volume of the sample is adjusted,
(B) obtaining two plates that are movable relative to each other in different configurations, one or two of the plates being flexible and one or two of said plates comprising spacers , The spacers have a predetermined distance and height between the spacers, and each spacer is fixed to its corresponding plate,
(C) depositing the sample on one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) after (c) the step of spreading the sample by placing the plate in a closed configuration, wherein the plates face each other in the closed configuration, and the spacer, the associated volume of sample and the height of the spacer One or more dusts of even greater thickness are located between the plates, and the thickness of the sample of associated volume is adjusted by the plates and spacers;
The spacer and the plate are configured to minimize the dust-affected area between the two plates, and in the dust-affected area, the dust-blocking spacer is similar to the plate without dust. Adjusting the final spacing between the plates in the region in the closed configuration of, the relevant volume being a portion of or the entire volume of the sample.

  In the method of paragraph U3, configuring the spacers and plates to minimize the area of influence of dust includes selecting an appropriate thickness and mechanical properties of the flexible plate.

  In some embodiments, by selecting the distance between the spacers, for allowed sample thickness variations, two plates given, and for the compression method, the curvature of the two plates due to the compression method is allowed. It is less than the fluctuation of the sample thickness. When in the closed configuration, the adjusted sample thickness can be less than the maximum thickness of the sample when the plate is in the open configuration.

  The flexibility of the plate is described.

U4. A device for minimizing the effect of dust on the thickness adjustment of a sample of relevant volume,
A first plate and a second plate that are movable relative to each other to form a different structure, each plate having a sample contacting surface for contacting a sample, and one or two of the plates being flexible. Two plates,
Comprising spacers on one or two sample contacting surfaces of the plate, each spacer having a predetermined distance and height between the spacers, each spacer being secured to a corresponding plate,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other and have a spacer, an associated volume of sample, and a thickness greater than the height of the spacer. One or more dusts are located between the plates, the thickness of the sample of the relevant volume is adjusted by the plates and spacers, and the spacers and plates are the area affected by the dusts between the two plates. The area affected by the dust, which is configured to minimize, is the area of the inner surface of the plate where the plate and spacers can no longer adjust the sample thickness like the dust-free area and is relevant. The volume of the device is a portion or the entire volume of the sample.

3.3 Use of spacers to reduce the effects of surface flatness variations In practice, no plate has a perfectly flat surface. As shown in FIG. 7a, in CROF, the variation in surface flatness can be very large compared to the desired sample thickness, which can cause a large error when determining the sample thickness. is there. The thinner the final sample in the CROF (for example, arranged in micrometers or nanometers), the variation in surface flatness can cause increasingly significant errors.

  The surface flatness variation is characterized by the plate's surface flatness variation distance, where λ is the distance from a local maximum in surface height to an adjacent local minimum (shown in Figure 7b).

  The present invention provides a method for making the final sample thickness variation in the closed configuration in the CROF process less than the surface flatness variation on the plate sample surface that is present when the plate is in the open configuration. In the present invention, an important way to achieve final sample thickness uniformity is to use flexible plates, proper spacer distance and proper pressure (shown in Figures 7c and 7d). ..

  Considering the use of one rigid plate and a flexible plate in the CROF process, the open configuration of the plate has good flatness on the sample surface of the rigid plate, but significant surface flatness on the sample surface of the flexible plate. It has a degree variation (i.e., is more pronounced compared to the expected final sample thickness) and is shown in Figures 7a and 7b. The present invention provides (i) a distance between spacers that is less than the initial surface flatness variation distance, (ii) an appropriate compressive force and/or an appropriate compressive force to deform the flexible plate between the sample and the plate in the closed configuration. In the final form of the plate, by calibrating the initial flatness variation of the sample surface in the open configuration (eg, reducing the flatness variation), by appropriate capillary force, (iii) proper flexibility of the flexible plate. , The sample surface of the flexible plate is deformed to match the contour of the flat surface of the rigid plate (Fig. 7c). Also, the distance between the spacers should be less than the critical bending span of the flexible plate to reduce variations in final sample thickness.

  The method of calibrating the variation of surface flatness is (b) flexible when the rigid plate has a significant initial sample surface flatness variation while the flexible plate has a smooth sample surface. If both the plate and the rigid plate have significant flatness variations on their expected sample surfaces, and (c) both plates are flexible and are noticeable on the sample surface of one or two plates. It also applies to the case with a large surface flatness variation (FIG. 7d).

U5. A method for reducing the effect of surface flatness variation of a plate on the final thickness uniformity of a sample of relevant volume in a CROF process, comprising:
(A) obtaining a sample, wherein the thickness of the relevant volume of the sample is adjusted,
(B) obtaining two plates that are movable relative to each other in different configurations, one or two of the plates being flexible and one or two of said plates being surface flat. Having a variation and one or two of said plates comprises spacers, said spacers having a predetermined height, each spacer being fixed to its corresponding plate,
(C) depositing the sample on one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) after (c) the step of spreading the sample by placing the plates in a closed configuration, wherein the plates face each other in the closed configuration and the spacer and the associated volume of sample are located between the plates. And the thickness of the relevant volume of sample is adjusted by plates and spacers,
The spacer and plate are configured such that the thickness variation of the relevant volume of the sample in the closed configuration is less than the surface flatness variation of the open configuration of the plate, the relevant volume being a fraction of the volume of the sample. Or the entire volume.

U6. An apparatus for reducing the effect of surface flatness variation of a plate on adjusting the thickness uniformity of a sample of related volume, which is movable relative to each other in different configurations, one of the plates or A first plate and a second plate, two of which are flexible and one or two of said plates have a surface flatness variation,
A spacer fixed on one or two of said plates and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration, which is constructed after the sample is deposited in an open configuration, in which the plates face each other and the spacer and associated volume of sample are located between the plates and are associated. The thickness of the volume sample is controlled by plates and spacers,
The spacer and plate are configured such that the thickness variation of the relevant volume of the sample in the closed configuration is less than the surface flatness variation of the open configuration of the plate, the relevant volume being a fraction of the volume of the sample. Or the entire volume, the average dimension of the relevant volume being greater than the surface flatness variation of the plate in the open configuration.

  In the method of paragraph U5 and the apparatus of paragraph U6, spacer and plate configurations that reduce the effect of plate surface flatness variations on the uniformity of the final thickness of the sample of the relevant volume are provided with suitable spacer spacing. Including using (IDS). In one preferred embodiment, the IDS is less than or equal to the plate's initial surface flatness variation distance in the open configuration.

  In the method and apparatus of paragraphs U5 and U6, in some embodiments, (1) the spacer is inside the sample in a closed configuration; (2) the spacer is fixed to a corresponding plate; ) Having a short distance between spacers, or (4) having any combination thereof.

  In the method and apparatus of paragraphs U1-U8, the above embodiments are included in the spacer and plate configurations that equalize the thickness of the relevant volume of the sample. In some embodiments, the distance between the given spacers is configured to limit the local curvature of the plate between the two spacers, and the relevant volume is a portion or the entire volume of the sample.

  This includes the case where one or two of the plates are flexible and of various different flexibility. (For example, PMMA or PS having a thickness of 100 μm).

  In one preferred embodiment, one plate is PMMA. In one preferred embodiment, one plate (first plate) is glass with a thickness of 0.5-1.5 mm and is not provided with spacers, the other plate (second plate) is A 175 μm thick PMMA film, and comprising an array of spacers, the spacers being rectangular columns with a circular angle (dimensions in the x direction of 40 μm and dimensions in the y direction of 30 μm), x The period has a period of 120 μm and the dimension in the y direction is 110 μm (therefore, the distance between the spacers is 80 μm in both the x direction and the y direction).

  In the method and apparatus of Section 3, in some embodiments of the spacer in the sample in the closed configuration, the spacer material and plate are those of Section 2.

  In the methods and apparatus of paragraphs U1-6, in some embodiments, the ratio of column width (or lateral average dimension) to column height is 0.01 or greater, 0.1 or greater, 1 or greater, 1 or greater. 0.5 or more, 2 or more, 3 or more, 5 or more, 10 or more, 50 or more, 100 or more, or a range between any two of these values.

  In the methods and apparatus of paragraphs U1-6, in a preferred embodiment, the ratio of column width (or lateral average dimension) to column height is 1, 1.5, 2, 10, 20, 30, 50, 100, or a range between any two of these values.

  In the methods and apparatus of paragraphs U1-6, in some embodiments, the ratio of the period of the columns to the width (or average lateral dimension) of the columns is 1.01, 1.1, 1.2, 1.5. , 1.7, 2, 3, 5, 7, 10, 20, 50, 100, 500, 1000, 10,000, or a range between any two of these values.

  In the methods and apparatus of paragraphs U1-6, in a preferred embodiment, the ratio of column period to column width (or lateral average dimension) is 1.2, 1.5, 1.7, 2, 3, 5. , 7, 10, 20, 30, or any two between these values.

  In the methods and apparatus of paragraphs U1-6, in a preferred embodiment, the ratio of column period to column width (or lateral average dimension) is 1.2, 1.5, 1.7, 2, 3, 5. , 7, 10, or a range between any two of these values.

  c) For example, for hemocytometry applications, the preferred X-plate column height is between 1 μm and 5 μm, the column width is between 2 μm and 30 μm, and the column period is between 4 μm and 300 μm.

  d) For example, in immunological assay applications, the column height of the preferred X-plate is 5 μm to 50 μm, the column width is 10 μm to 250 μm, and the column period is 20 μm to 2500 μm.

  The embodiments described in Section 3 and any combination thereof apply to (ie are combined with) other embodiments throughout the description of the invention.

  In some embodiments, other factors are also used to control sample thickness uniformity, such factors as sample area, plate mechanical properties, final sample thickness under closed configuration and plate surface wetting properties. Including but not limited to.

  Below are some preferred embodiments of the method and apparatus in Section 1 and other disclosures.

  In one preferred embodiment, the spacers are periodic square arrays, of which the spacers have a height of 2 μm to 4 μm, an average lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 1 μm to 100 μm. It has a columnar shape.

  In one preferred embodiment, the spacers are periodic square arrays, the spacers having a height of 2 μm to 4 μm, a mean lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 100 μm to 250 μm. belongs to.

  In one preferred embodiment, the spacers are periodic square arrays, the spacers having a height of 4 μm to 50 μm, a mean lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 1 μm to 100 μm. belongs to.

  In one preferred embodiment, the spacers are periodic square arrays, of which the spacers have a height of 4 μm to 50 μm, an average lateral dimension of 5 μm to 20 μm, and a spacing between the spacers of 100 μm to 250 μm. belongs to.

  The period of the spacer array is in one preferred embodiment between 1 nm and 100 nm, in another preferred embodiment between 100 nm and 500 nm, and in one single preferred embodiment between 500 nm and 1000 nm. And in another preferred embodiment between 1 μm (ie 1000 nm) and 2 μm, in another preferred embodiment between 2 μm and 3 μm, in another preferred embodiment between 3 μm and 5 μm. In another preferred embodiment between 5 μm and 10 μm, in another preferred embodiment between 10 μm and 50 μm, in one single preferred embodiment between 50 μm and 100 μm, In one single preferred embodiment, between 100 μm and 175 μm, and in one single preferred embodiment, between 175 μm and 300 μm.

4. Sample and deposition In the method and apparatus of the present invention using the CROF process, the sample is deposited by several methods or a combination of these methods. In one particular embodiment of attachment, the sample attaches to only one plate. In certain embodiments, the sample adheres to two plates (ie, the first plate and the second plate).

  The sample adheres when the plate is in the open configuration. In some embodiments, the first plate and the second plate are separated from each other during sample deposition, which allows the sample to adhere to one plate and not interfere with another plate. For example, the first plate and the second plate may be far apart, which allows the sample to fall directly onto the first plate or the second plate and the absence of another plate. .. In certain embodiments of sample deposition, the first plate and the second plate are spaced a distance apart from each other under the open configuration of the plate, and then the sample is deposited on the plate (eg, horizontal flow or other drip). By method). In a particular embodiment, the two plates have one side (eg edge) (FIG. 30) connected to them during all operating steps of the plate, opening and closing the two plates is similar to opening and closing a book. ..

  The sample attachment may be a single drop or multiple drops. Multiple drops can fall on one plate or on two plates at one or more positions. The droplets can be well separated from each other, connected, or combined.

  In some embodiments, the sample comprises one or more materials, said materials being attached together or separately. The materials are deposited in parallel or sequentially.

  The attachment of the sample to the plates (ie the first plate and the second plate) may be done using the device or directly from the subject to the plates. In some embodiments, the device is used to deposit the sample. Devices include, but are not limited to, pipettes, needles, sticks, swabs, tubes, ejectors, liquid dispensers, tips, sticks, inkjets, printers, ejectors and the like. In certain embodiments, the sample is attached by direct contact between the sample of the sample source and the CROF plate, without the use of any equipment (ie, the sample and plate are brought into contact with each other). . This is referred to by the term "direct sample attachment".

  Examples of direct sample attachment to the plate are: (a) direct contact between the punctured finger (or other body part) and the plate, (b) exhaling saliva onto the plate. , (C) removing the tear fluid from the eye by direct contact between the tear fluid and the plate, (d) direct contact between the sweat fluid and the plate, and (e) exhaling directly by exhaling breath to the plate. Etc. Such direct attachment methods should be applicable to humans and animals.

  In some embodiments, both direct and indirect (by device) sample attachment are used.

  In the present invention, the volume of the sample adhered to one plate or a plurality of plates (“sample volume”) is 0.001 pL (picoliter) at maximum, 0.01 pL at maximum, 0.1 pL at maximum, and 1 pL at maximum. , Maximum 10 pL, maximum 100 pL, maximum 1 nL (nanoliter), maximum 10 nL, maximum 100 nL, maximum 1 μL (microliter), maximum 10 μL, maximum 100 μL, maximum 1 mL (milliliter), maximum 10 mL or these Is a range between any two values of.

  In some embodiments, sample attachment involves sample deposition using a scheme other than (a) placing the sample on one or two plates, and (b) compressing a second plate in the CROF process. Including the step of unfolding. Methods of spreading the sample include using another device (eg, stick, cutter), blowing or otherwise.

Sample Deformation During the CROF process, in some embodiments, the sample behaves almost like an incompressible liquid (referring to a liquid that holds a constant volume under shape deformation), so that a change in sample thickness causes a change in sample area. Cause a change. In some embodiments, the sample behaves like a compressed liquid, but the side area still expands as its thickness decreases during the CROF process. In certain embodiments, the sample is a liquid, gel or soft solid, and may have an increased side area as its thickness decreases during the CROF process.

  In the disclosure of the present invention, "opposing the first plate and the second plate" means that the sample is moved to the first plate and the second plate by manipulating the position and the direction of the first plate or the second plate or both. Is the process of being between the inner surfaces of the plates. In some embodiments, the act of "facing the first plate and the second plate" is performed by a human hand, a human hand with a particular device, or an automated device without a human hand.

  In some embodiments, the thickness is up to 1 mm, up to 100 μm, up to 20 μm, up to 10 μm or up to 2 μm. In some embodiments, the thickness is at least 0.1 μm. In some embodiments, further including thickness measurement.

  In some embodiments, the variation in thickness of the relevant volume of the sample is at most 300%, at most 100%, at most 30%, at most 10% of the effective diameter of the relevant region. %, at most 3%, at most 1%, at most 0.3%, or at most 0.1%.

  In some embodiments, the thickness is at least partially determined by the preset height.

5. Analytes, Entities, Binding Sites, Storage Sites, and Transport Vehicles In the present invention, entities include but are not limited to one of proteins, amino acids, nucleic acids, lipids, carbohydrates, metabolites, cells or nanoparticles. There is no such thing.

  In some embodiments, the binding site comprises a binding partner configured to bind the entity.

  In some embodiments, the binding site comprises an entity attached to the binding site.

  In some embodiments, placing the sample comprises placing the sample within the binding site.

  In some embodiments, the reagents include at least one of proteins, amino acids, nucleic acids, lipids, carbohydrates and metabolites.

  In certain embodiments, the storage site contains dry reagents.

  In some embodiments, the storage site comprises a reagent configured to be released from the storage site upon contact with the sample.

  In some embodiments, the first storage site and the second storage site are located in one joint storage site.

  In some embodiments, the transfer medium is a sample. In some embodiments, the transfer medium is a liquid, where the reagent or entity can dissolve and diffuse in the liquid.

  In some embodiments, the plate has multiple storage sites. In another embodiment, one reservoir has various reagents.

  Different Release Times In some embodiments, the plate has multiple storage sites at different locations on the plate or stores multiple reagents in one storage site, the reagents being released upon contact with the sample at the storage sites, but Different reagents from the same storage site or reagents from different storage sites are released at different times.

  In some embodiments, the first reagent is arranged to be released from the first storage site within a first average release time upon contact with the sample, and the second reagent is disposed on the second contact upon contact with the sample. Arranged to be released from the second storage site within an average release time, wherein the first average release time is less than the second average release time.

  In some embodiments, the first reagent is arranged to be released from the first storage site upon contact with the sample, wherein the second reagent is the binding reagent.

  In some embodiments, attaching comprises binding at least one reagent to the corresponding plate.

  In some embodiments, contacting comprises releasing at least one reagent from the corresponding plate.

  In some embodiments, depositing comprises depositing a first reagent and a second reagent, wherein contacting comprises releasing the first reagent prior to the second reagent.

  In some embodiments, at least one plate contains a reservoir, which contains reagents that are added to the relevant volume of the sample.

  In some embodiments, the reagent comprises at least one of proteins, amino acids, nucleic acids, lipids, carbohydrates and metabolites.

  In some embodiments, the storage site comprises dry reagents.

  In some embodiments, the storage site comprises a reagent configured to be released from the storage site upon contact with the sample.

  In some embodiments, the reservoir is the first reservoir and the reagent is the first reagent, where the device comprises a second reservoir, which is added to the relevant volume of the sample. A second reagent, wherein the second storage site is located on one of the plates.

  In some embodiments, the first storage site and the second storage site are located in one joint storage site.

  In some embodiments, the first reagent is arranged to be released from the first storage site within a first average release time upon contact with the sample, and the second reagent is disposed on the second contact upon contact with the sample. Arranged to be released from the second storage site within an average release time, the first average release time being less than the second average release time.

  In some embodiments, at least one reagent is dried on the corresponding plate.

  In some embodiments of the kit, at least one reagent is bound to the corresponding plate.

  In some embodiments of the kit, the at least one reagent is arranged to be released from the corresponding plate upon contact with the sample.

  In some embodiments of the kit, the first reagent is located on one or two of the plates and the second reagent is located on one or two of the plates, where the first reagent is on the sample. The second reagent is arranged to be released from the corresponding plate within a first average release time upon contact, and the second reagent is arranged to be released from the corresponding plate within a second average release time upon contact with the sample. , Where the first average release time is less than the second average release time.

  In some embodiments of the device, the reservoir is the first reservoir and the reagent is the first reagent and the device comprises the second reservoir, which is added to the relevant volume of the sample. A second storage site, the second storage site being located on one of the plates.

6. Local binding or mixing (P) in some samples
In some applications, it is desirable to have a binding site that captures (ie, binds) the analyte in some of the samples rather than the analyte in the entire sample. In some cases it is desirable to add (ie, mix) the reagents to a portion of the sample rather than the entire sample. It is generally desirable that there be no fluid separation between one part of the sample and the other part of the sample. In multiplex detection, such a requirement is preferable or necessary.

  The present invention uses the CROF method and apparatus to reform a sample into an ultrathin film whose thickness is less than the lateral dimension of a portion of the sample, thereby providing a solution to the above requirements, where the sample Only the analyte inside the compartment is captured or only a portion of the sample mixes with the reagent. The working principle of such an approach is as follows: when the sample thickness is smaller than the lateral dimension of some samples, the capture of the analyte by the surface or the mixing of the reagents arranged on the surface is mainly analyzed. The diffusion of objects and reagents in the thickness direction is limited, where the diffusion of horizontal diffusion is relatively insignificant. For example, if the sample is reformed into a thin film of 5 μm thickness, if some of the analytes in the sample that need to be captured or reagents that need to be mixed have a lateral dimension of 5 mm×5 mm, then And if the diffusion time of the analyte or reagent over 5 μm is 10 seconds, the horizontal diffusion of the analyte or reagent over the 5 mm distance is 1,000,000 seconds (because the diffusion time is directly proportional to the square of the diffusion distance). is there. This is because, by choosing an appropriate ratio of the lateral dimension and the sample thickness of the portion of interest of the sample over a fixed time interval, the analyte to be captured is mainly derived from the sample portion of interest. Alternatively, the reagents are primarily mixed with the sample portion of interest.

6.1 Local bond between the surface of a part of the sample and the surface (P: volume to surface)
P1. A method for locally binding a target entity in a sample of relevant volume to a binding site on a surface, comprising:
(I) Performing steps (a)-(d) in the method of paragraph X1, wherein the sample thickness in the closed configuration is significantly less than the average linear dimension of the binding site and the relevant volume is the plate closed. The sample volume located at the binding site when in the configuration, step,
(Ii) After (i), while the plate is in the closed configuration,
(1) incubating the sample for a relevant length of time and then stopping the incubation, or
(2) Incubating the sample for a time greater than or equal to the minimum relevant time length and then assessing the binding of the target entity to the binding site within a time less than or equal to the maximum relevant time length. And
The relevant length of time is
i. More than the time required for the target entity to diffuse through the thickness of the layer of uniform thickness in the closed configuration, and ii. Significantly shorter than the time required for the target entity to spread laterally across the smallest horizontal dimension of the binding site,
At the end of the incubation in (1) or during the evaluation in (2), most of the target entity bound to the binding site comes from a relevant volume of sample,
Incubation allows the target entity to bind to the binding site, the relevant volume being a part of the sample at the binding site in the closed configuration, the method comprising the steps.

  In the method of paragraph P2, the term "the thickness of the sample of the relevant volume is significantly less than the minimum average size of the binding sites" means the ratio of the minimum average size of the binding sites to the sample thickness ("length to thickness"). "Ratio") is at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, at least 1,000, at least 10,000, at least 100,000, or any value between these. It is in the range of. In a preferred embodiment, the length to thickness ratio is at least 3, at least 5, at least 10, at least 20, at least 50, at least 100, at least 500, or any range between these values.

  In the method of paragraph P2, the term "significantly shorter than the time required for the target entity to diffuse over the minimum lateral dimension of the binding site" refers to the minimum lateral extent of the binding site with respect to the time required for diffusion over the sample thickness. The ratio of the time required to diffuse over the directional dimension (called the "length ratio to thickness diffusion time") is at least 3, at least 10, at least 50, at least 10, at least 100, at least 1,000, at least 10, 000, a minimum of 100,000, a minimum of 100,000, or any range between these values. In a preferred embodiment, the ratio of length to thickness diffusion time is at least 3, at least 10, at least 50, at least 10, at least 100, at least 1,000, at least 10,000, or any range between these values. Is.

P2. A device for locally binding an entity in a sample of a relevant volume to a binding site on a surface,
Including a first plate and a second plate movable relative to each other in a different configuration;
The first plate has a binding site on its surface having an area smaller than the area of the plate and is configured to bind to a target entity in the sample, the target entity being able to diffuse into the sample, One or two each include a spacer, each spacer being fixed to its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration configured after sample attachment in an open configuration, and in the closed configuration, the plates face each other and the spacers, binding sites, and at least some of the sample are located between the plates, The sample is in contact with at least a portion of the binding site, and the thickness of the relevant volume of the sample is adjusted by the plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration, The relevant volume is the volume located at the binding site of the sample,
The height of the spacers is selected to adjust the thickness of the relevant volume under the closed configuration to be less than at least one third of the average linear dimension of the binding site.

  By adjusting the thickness of the relevant volume to one-third of the average linear dimension of the binding site, the diffusion time over the sample thickness of the entity is 9 times less than the diffusion time over a distance equal to the average linear dimension of the binding site. It is 1. Such thickness control allows for the selection of incubation times whereby (i) a significant number of target entities in the relevant volume are bound to the binding site and (ii) to the binding site. The incubation result is that a significant amount of the target entity is derived from a relevant volume of the sample, which is a process that allows the target entity to bind to the binding site.

  For example, if the incubation time is set equal to the diffusion time over the thickness of the relevant volume of the sample, after incubation most of the entities within the relevant volume have already reached the binding site, and Entities bound according to the rate equation and located outside the relevant volume initially (ie prior to incubation) can diffuse only around the relevant volume (relatively small volume) and are associated with the mean linear dimension of the binding site. The importance of this volume is diminished as the ratio to a certain volume thickness is increased.

6.2 Local binding of entities stored on a plate surface to binding sites on another plate surface (from surface to another surface)
P3. A method for locally binding an entity stored on a storage site of one plate to a binding site on another plate, the method comprising:
(A) obtaining a first plate and a second plate that are movable relative to each other in different configurations, the first plate having a binding site on its surface, The plate has on its surface a storage site containing an entity that binds to the binding site, the area of said binding site and the area of the reagent site being smaller than the area of its corresponding plate, and one or two of said plates Spacers, each spacer being secured to its corresponding plate and having a predetermined height;
(B) obtaining a transport medium in which the entity is soluble and diffusible in the transport medium,
(C) depositing a transfer medium onto one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated. And the spacing between the plates is not adjusted by the spacer, a step,
(D) after (c) expanding the transfer medium in a closed configuration with the plates facing each other in the closed configuration, the spacer, the binding site, the storage site, and at least a portion of the transfer medium. Are located between the plates, at least some of the storage sites face the binding sites, have some of the transport medium therebetween, and the thickness of the relevant volume of transport medium is adjusted by the plates and spacers. The thickness is less than the maximum thickness of the sample when the plate is in the open configuration, and the thickness is significantly less than the average linear dimension of the relevant volume along the surface of the plate. And (e) after (d) the step of incubating the sample for a period of time and stopping the incubation while the plate is in the closed configuration, wherein a substantial number of entities bound to the binding site are The incubation time is selected to be from, the volume of interest is the volume of the transfer medium at the binding site, and the incubation is a process that allows binding of the entity to the binding site, including steps. ,Method.

  The term "at least some storage sites face a binding site" refers to the shortest distance from a point on the site to the binding site being the same as the relevant volume thickness in the closed configuration of the plate. ..

P4. A device for binding an entity stored on a storage site of one plate to an associated binding site on another plate, comprising:
Movable relative to each other in different configurations, the first plate has a binding site on its surface, and the second plate has a storage site on its surface containing an entity that binds to the binding site. The area of the binding site and the area of the storage site is smaller than the area of its respective plate, and one or two of said plates comprises spacers, each spacer being fixed to its respective plate and having a predetermined A first plate and a second plate having a height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the transfer medium adheres to one or two of said plates, The entity above is capable of dissolving in the transport medium and diffusing into the transport medium,
Another configuration is a closed configuration that is configured after the transfer medium is deposited in the open configuration, wherein the plates face each other and the spacer, the binding site, the storage site, and at least some of the transfer medium are in the closed configuration. Located between the plates, at least some of the storage sites facing the binding sites and having some of the transport medium therebetween, the thickness of the relevant volume of transport medium is adjusted by the plates and spacers, And said thickness is less than the maximum thickness of the sample when the plate is in the open configuration,
The volume of interest is the volume of transfer medium that is on the storage site when the plate is in the closed configuration, and the height of the spacer is the thickness of the volume of interest in the closed configuration of the average linear dimension of the binding site. Selected to adjust to at least less than one-third,
At least one spacer is located in the sample contact area,
And, the spacer has a predetermined distance and height between the spacers.

6.3 Method for locally binding entities on multiple storage sites on one plate to multiple corresponding binding sites on another plate P5. A method for locally binding an entity stored on a plurality of storage sites on one plate to a plurality of corresponding binding sites on another plate, the method comprising:
(A) obtaining a first plate and a second plate that are movable relative to each other so as to have different configurations, wherein the surface of the first plate has a plurality of binding sites, The surface of the second plate has a plurality of corresponding storage sites, and each corresponding storage site is bonded such that each binding site overlaps only one storage site when the two plates are arranged facing each other. Located at a position of the second plate corresponding to the position of the site, one or two of the plates including spacers, each spacer being fixed to its corresponding plate and having a predetermined height;
(B) obtaining a transfer medium, wherein the entity at the storage site is capable of dissolving and diffusing in the transfer medium,
(C) depositing a transfer medium onto one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated. And the spacing between the plates is not adjusted by the spacer, a step,
(D) After (c) the step of spreading the transfer medium in the closed configuration of the plates, wherein the two plates face each other in the closed configuration, the spacer, the binding site, the storage site, and at least a portion of the transfer A medium is located between the plates, each binding site faces only one corresponding storage site, and a transfer medium contacts at least a portion of each binding site and a portion of each storage site, The thickness of the volume of transfer medium is adjusted by the plates and spacers, the thickness being less than the maximum thickness of the transfer medium when the plate is in the open configuration and significantly less than the average linear dimension of the binding site, After steps, and (e)(d), incubating the sample for a period of time and stopping the incubation while the plate is in the closed configuration, wherein a substantial number of entities bound to each binding site are present. , The incubation time is chosen to be from the corresponding storage site, the relevant volume is the volume of the transfer medium at the binding site, and the incubation is a process that allows binding of the entity to the binding site. A method comprising the steps of:

  In some embodiments, the spacing is limited to the sample binding area.

  In some embodiments of Method P5, the transfer medium is a sample having a target analyte, the binding site comprises a capture agent, the entity at the storage site is a detection agent, and the target analyte is a capture agent-analyte. It binds to the capture and detection agents to form an object-detection agent sandwich. Method P5 simplifies the assay step and shortens the assay time by using smaller spacer heights, resulting in a thinner sample thickness for both analyte and detection agent for shorter saturation assay times. And shorter vertical diffusion times.

P6. A device for locally binding an entity stored on a plurality of storage sites on one plate to a plurality of corresponding binding sites on another plate,
A first plate and a second plate movable relative to each other in different configurations,
The surface of the first plate has a plurality of binding sites and the surface of the second plate has a plurality of corresponding storage sites, each binding site when the two plates are arranged facing each other. Each corresponding storage site is located at a position on the second plate corresponding to the position of the binding site, such that one overlaps with only one storage site, one or two of said plates including spacers, and each spacer Is fixed to its corresponding plate and has a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the transfer medium adheres to one or two of said plates, the storage site The above entity can dissolve in the transport medium and diffuse into the transport medium,
Another configuration is a closed configuration that is configured after the transfer medium is deposited in the open configuration, in which the two plates face each other and the spacer, the binding site, the storage site, and at least a portion of the transfer medium are Located between the plates, each binding site faces only one corresponding storage site, and the transfer medium contacts and is associated with at least a portion of each binding site and a portion of each storage site. The volume of the transport medium is adjusted by plates and spacers, the thickness being less than the maximum thickness of the transport medium when the plate is in the open configuration,
The volume of interest is the volume of transfer medium at the storage site when the plate is in the closed configuration, and the predetermined spacer height is selected to adjust the thickness of the volume of interest in the open configuration. , A device significantly smaller than the average linear dimension of the binding site.

6.4 Local addition of surface-stored reagents to a portion of the sample (surface to volume)
P7. A method for locally adding a reagent into a sample of relevant volume, comprising:
(A) Obtaining a first plate and a second plate that are movable relative to each other in different configurations, the first plate being added to a volume of sample associated with its surface. Having a storage site for the reagent, the reagent being capable of dissolving in the sample and diffusing into the sample, and having an area of the storage site smaller than the area of the plate, one or two of said plates comprising spacers, and A spacer, fixed to its corresponding plate and having a predetermined height, a step,
(B) obtaining a sample,
(C) depositing a sample on one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and A configuration in which the spacing between the plates is not adjusted by spacers, steps,
(D) after (c) the step of spreading the sample in the plate in a closed configuration, wherein the plates face each other and the spacers, storage sites and at least a portion of the sample are in the plate. And the area of contact of the sample with at least a portion of the storage site and with the plate is greater than the area of contact with the storage site and the thickness of the sample of the relevant volume is between the plate and A step adjusted by a spacer such that the thickness is less than the maximum thickness of the sample when the plate is in the open configuration and is significantly less than the average linear dimension of the relevant volume in the surface direction of the plate; e) after (d), incubating the sample for a certain period of time and stopping the incubation while the plate is in the closed configuration, wherein (i) a large volume of reagent dissolved in the sample has a relevant volume. Incubation time is selected such that it is included in the sample of (1) and (ii) the reagent is at the prominent site of the relevant volume, and the relevant volume is at the binding site when the plate is in the closed configuration. A method comprising the steps of: being a volume of a sample, wherein incubation is a process that allows binding of an entity to a binding site.

P8. Device for locally adding a reagent stored on the surface of a plate to a sample of related volume, comprising a first plate and a second plate movable relative to each other in different configurations ,
The first plate has a storage site for a reagent that is added to the volume of the sample associated with its surface, the reagent being capable of dissolving in the sample and diffusing into the sample, one or two of the plates. One includes a spacer, and each spacer is fixed to its corresponding plate and has a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration that is configured after the sample is deposited in the open configuration, where the plates face each other and the spacer, the storage site, and at least a portion of the sample are located between the plates. The sample contacts at least a portion of the storage site and at least a portion of the plate surface other than the storage site, and the thickness of the relevant volume of the sample is adjusted by the plate and the spacer, the thickness being such that the plate is open. Less than the maximum thickness of the sample when in the configuration, the relevant volume is the volume of the sample at the storage site when the plate is in the closed configuration,
The apparatus wherein the predetermined spacer height is selected to adjust the thickness of the relevant volume in the open configuration of the plate and is three times less than the average linear dimension of the relevant volume in the surface direction of the plate.

7. Formation of a capture agent-analyte-detection agent sandwich on the binding site (W)
One aspect of the invention is that the CROF process places binding sites on a solid surface by placing the binding sites on one plate and the storage sites for storing the detection agent at corresponding positions on another plate. In one step, a capture agent-analyte-detection agent sandwich is formed.

7.1 Formation of a capture agent-analyte-detection agent sandwich on the binding site in one step of incubation (general) (W)
W1. A method for forming a capture agent-analyte-detection agent sandwich on a binding site of a plate, comprising:
(A) obtaining a sample containing a target analyte that can diffuse into the sample,
(B) obtaining a capture agent and a detection agent, wherein the capture agent and the detection agent bind (are capable of binding) to the target analyte to form a capture agent-target analyte-detection agent sandwich. , Step,
(C) a step of obtaining a first plate and a second plate that are movable with respect to each other so as to have different configurations, wherein the first plate has a binding site on which a capture agent is immobilized. And the second plate has a storage site for storing the detection agent, the detection agent being capable of dissolving in the sample and diffusing into the sample upon contact of the storage site with the sample, one or two of the plates One of which includes a spacer, and each spacer is fixed to its corresponding plate and has a predetermined height,
(D) depositing a sample on one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and A configuration in which the spacing between is not adjusted by spacers, and (e) after (d) spreading the sample in the closed configuration to spread the sample, the plates facing each other in the closed configuration. A spacer and an associated volume of sample are located between the plates, and the thickness of the associated volume of sample is adjusted by the plate and the spacer, the thickness being when the plate is in the open configuration. Thinner than the thickness of the sample, the sample contacting the binding and storage sites,
(F) after (e), incubating the sample while the plate is in the closed configuration for a time that allows formation of the capture agent-target analyte-detection agent sandwich,
The method wherein the relevant volume is at least a portion or the total volume of the sample.

W2. A device for forming a capture agent-analyte-detection agent sandwich on a binding site of a plate, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a binding site on which the capture agent is immobilized, the second plate has a storage site for storing the detection agent, and the capture agent and the detection agent have a capture agent-target assay. An analyte-detection agent that binds (is capable of binding) a target analyte in the sample to form a sandwich and the storage site contacts the sample so that the detection agent can dissolve in the sample and diffuse into the sample; One or two of which include spacers, and each spacer is fixed to its corresponding plate and has a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration configured after the sample is deposited in the open configuration, wherein the plates face each other and a volume of sample associated with the spacer is located between the plates. The thickness of the relevant volume of sample is adjusted by the plate and the spacer, the thickness being less than the thickness of the sample when the plate is in the open configuration, the sample being in contact with the binding site and the storage site,
The apparatus wherein the relevant volume is at least a portion or total volume of the sample.

7.2 Formation of Capture Agent-Analyte-Detector Sandwich on Binding Site in Single Step Incubation with Analyte from a Part of the Sample (ie Topically) W3. A method for forming a capture agent-analyte-detection agent sandwich on a binding site of a plate using an analyte from a portion of a sample, comprising:
(A) obtaining a sample containing a target analyte that can diffuse into the sample,
(B) obtaining a capture agent and a detection agent, wherein the capture agent and the detection agent bind to the target analyte to form a capture agent-target analyte-detection agent sandwich.
(C) a step of obtaining a first plate and a second plate that are movable with respect to each other so as to have different configurations, wherein the first plate has a binding site on which a capture agent is immobilized. And the second plate has a storage site for storing the detection agent, the detection agent being capable of dissolving in the sample and diffusing into the sample upon contact of the reagent storage site with the sample, one of the plates or Two including spacers, each spacer being fixed to its corresponding plate and having a predetermined height;
(D) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and the plates are The spacing between is not adjusted by the spacer, is a step,
(E) after (d) spreading the sample in a plate in a closed configuration, wherein the plates face each other and the spacer, the binding site and the storage site are located between the plates. The binding site and the storage site are in contact with the relevant volume of the sample, and the thickness of the sample of the product of association is adjusted by the plates and spacers, the thickness being the sample when the plate is in the open configuration. Less than the thickness of, and significantly smaller than the average linear dimension of the binding site, and (f) after (e), incubating the sample for a period of time and stopping the incubation while the plate is in the closed configuration. Where the incubation time is selected such that a significant number of capture agent-analyte-detector sandwiches formed at the binding site contain the analyte from the relevant volume of sample, and the relevant volume is the binding site. Volume of the sample at, and incubation is a process that allows the formation of a capture agent-analyte-detection agent sandwich.

  In some embodiments, the ratio of spacing to site size may be less than 1/5.

W4. An apparatus for forming a capture agent-analyte-detection agent sandwich on a binding site of a plate using an analyte from a portion of a sample, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a binding site on which a capture agent is immobilized, the second plate has a storage site for storing a detection agent, and the capture agent and the detection agent are a capture agent- It binds (is capable of binding) to the target analyte in the sample so as to form a target analyte-detection agent sandwich, and upon contact of the storage site with the sample the detection agent can dissolve in the sample and diffuse into the sample. One or two of said plates include spacers, each spacer being fixed to its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, wherein the plates face each other and the spacer, the binding site, and the storage site are located between the plates, and the binding The site and the storage site are in contact with the relevant volume of the sample, and the thickness of the relevant volume of the sample is adjusted by the plate and the spacer, the thickness of the sample being when the plate is in the open configuration. Less than the thickness, the relevant volume is the volume of the sample at the binding site, and the height of the spacer is chosen to control the thickness of the relevant volume in the closed configuration to determine the average binding site. A device that is significantly smaller than its linear dimensions.

7.3 Method for shortening the time to form a capture agent-analyte-detection agent sandwich on the binding site by reducing the diffusion distance (W, X)
W5. A method for reducing the time to form a capture agent-analyte-detection agent sandwich on a binding site of a plate, comprising:
(A) obtaining a sample containing a target analyte that can diffuse into the sample,
(B) obtaining a capture agent and a detection agent, wherein the capture agent and the detection agent bind to the target analyte to form a capture agent-target analyte-detection agent sandwich.
(C) a step of obtaining a first plate and a second plate that are movable with respect to each other so as to have different configurations, wherein the first plate has a binding site on which a capture agent is immobilized. The second plate has a storage site for storing the detection agent, and when the reagent storage site comes into contact with the sample, the detection agent can dissolve in the sample and diffuse into the sample. Or two including spacers, each spacer being fixed to its corresponding plate and having a predetermined height,
(D) depositing a sample on one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and The spacing between is not adjusted by the spacers, is a step,
(E) after (d) a step of spreading the sample in the plate in a closed configuration, wherein the plates face each other and have spacers, binding sites and storage sites in the closed configuration. Located between the plates, the binding site overlaps the storage site, the binding site and the storage site are in contact with the sample of the relevant volume, and the thickness of the sample of the relevant volume is adjusted by the plate and the spacer. Is less than the thickness of the sample when the plate is in the open configuration, thereby reducing the sample thickness and thus reducing the time for analyte and detection agent to diffuse through the thickness of the sample. , The relevant volume is at least a portion of the total volume of the sample, including the steps:
The method wherein the time for allowing the target entity in the relevant volume to bind to the binding site is shorter than the time without the closed configuration.

  The method further comprises a washing step that removes the sample between the plates and is performed when the plates are in the closed or open configuration.

  The method further comprises reading the signal from a capture agent-analyte-detection agent sandwich immobilized at the binding site. Read after or without any wash.

  The method may be further multiplexed, as described above or below.

W6. An apparatus for reducing the time to form a capture agent-analyte-detection agent sandwich on a binding site of a plate, the apparatus comprising a first plate and a second plate movable relative to each other in different configurations. Including,
The first plate has a binding site on which a capture agent is immobilized, the second plate has a storage site for storing a detection agent, and the capture agent and the detection agent are a capture agent-target. Binding (capable of binding) a target analyte in the sample to form an analyte-detection agent sandwich, and upon contact of said storage site with the sample, the detection agent can dissolve in the sample and diffuse into the sample; One or two of said plates include spacers, each spacer being fixed to its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, wherein the plates face each other and the spacer, the binding site, and the storage site are located between the plates, and the binding The site overlaps the storage site, the binding site and the storage site contact the relevant volume of the sample, and the thickness of the relevant volume of the sample is adjusted by the plate and the spacer, the thickness being Less than the thickness of the sample when in the open configuration, so that the time for the analyte and detection agent to diffuse vertically through the thickness of the sample is shortened by reducing the sample thickness and is relevant. The device wherein the volume is at least a portion of the total volume of the sample.

  In these embodiments, the method may include the step of attaching the capture agent to the plate by a chemical reaction between the capture agent and the reactive groups on the plate. Another plate may contain one detection reagent that has been dried in position so that the attached capture agent and detection reagent face each other after the plate is closed. Then, as described above, the method may include contacting a sample containing the target analyte with the device and closing the plate. The detection agent dissolves and diffuses in the sample. Since the target analyte is in solution, it is bound by the capture agent and immobilized on one surface of the plate. The detection reagent can bind to the target analyte before or after binding to the capture agent. In some cases, the method removes any target analyte that is not bound to the capture agent or all detection reagent that is not bound (eg, by washing the surface of the plate in binding buffer). Steps may be included and the detection agent may be coupled with an optically detectable label, thereby providing a method of detecting the target analyte. After optionally removing the detection agent not bound to the target analyte, for example, using a reading system to read the optical signal (eg, light in the wavelength range 300 nm to 1200 nm) from the detection agent bound to the plate. , The system can be read. Further, as mentioned above, the detection agent may be directly labeled (wherein the detection agent may be intimately connected with the luminescent label prior to attachment to one plate) or indirectly. It may be labeled (ie by means of a bound detection agent and a secondary capture agent, eg a labeled secondary antibody or a labeled nucleic acid, the secondary capture agent is specifically bound to the detection agent and attached to a luminescent label. ). In some embodiments, the method may include a blocking agent, thereby preventing non-specific binding between the capture agent and non-target analytes. Suitable conditions for specific binding of the target analyte to other reagents include suitable temperature, time, solution pH value, ambient light intensity, humidity, chemical reagent concentration, antigen-antibody ratio, etc., all of which are It is either well known or easily obtained from the present disclosure. General methods of molecular interaction between a capture agent and its binding partner, including the analyte, are well known in the art (eg, Harlow et al., Antibody: A Laboratory Manual, 1st Edition (1988). ) Cold spring Harbor, NY, Ausubel et al., Short Protocols in Molecular Biology, Third Edition, Wiley & Sons, 1995). The above and below methods are exemplary and the method herein is not the only way to perform an assay.

  In certain embodiments, nucleic acid capture agents can be used to capture protein analytes (eg, DNA or RNA binding proteins). In an alternative embodiment, protein capture agents (eg, DNA or RNA binding proteins) can be used to capture nucleic acid analytes.

  The sample may be a clinical sample from cells, tissues or body fluids. The target body fluids are amniotic fluid, aqueous humor, vitreous humor, blood (eg, whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax, chyle, chyme, Endolymph, perilymph, feces, stomach acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, purulent fluid, mucous secretions, saliva, sebum (skin oil), semen , Sputum, sweat, synovial fluid, tears, vomiting, urine and exhaled breath condensate.

  In one embodiment of the assay, the plate is contacted with a sample containing the target analyte (eg, target protein), followed by closing the plate. The sample contains or is modified to contain all necessary reagent (eg, salt, etc.) conditions suitable for specific binding. The capture agent (eg, antibody) and detection agent are capable of specifically binding to the target analyte in the sample, thereby detecting one labeled analyte.

  As with any embodiment, the amount of target analyte in the sample can be measured to provide a qualitative or quantitative measurement of the amount of target analyte in the sample. In some embodiments, the magnitude of the signal provides a quantitative measure of the amount of target analyte in the sample. In some cases, the evaluation can be compared to a standard curve (eg, a standard curve for a second analyte or spike-in analyte) that may be of known concentration in certain cases. The comparison can be facilitated by attaching capture agents at different densities (eg, different concentrations) and reading the signal from each capture agent.

8. Binding and addition of small volumes of sample and reagents (V)
In many applications it is highly desirable to use the smallest possible volume of sample or reagent. However, in microfluidic channel devices (currently the most popular devices that use small samples), the volume of the test location is wasted because a large amount of sample is wasted during the flow from the device inlet to the test (detection) area. Requires a larger sample volume. One aspect of the invention is used during testing by depositing a small volume of sample or reagent on a plate and then reshaping the volume into a thin film having a small thickness but a larger area than before. A significant reduction in sample or reagent volume. Such reformation makes the reaction faster.

8-1 Binding of target entity in a small volume of sample on the surface binding site by spreading the sample V1. A method for binding a target entity in a sample to a binding site, the method comprising:
(A) Obtaining a first plate and a second plate that are movable relative to each other in different configurations, the first plate having a binding site on its surface, One or two comprising spacers each fixed to its corresponding plate and having a predetermined height;
(B) obtaining a sample containing the target entity bound to the binding site,
(C) depositing a sample on one or two of the plates when the plates are configured in an open configuration, wherein the two plates are partially or completely separated, The spacing between is not adjusted by spacers, and the attached sample does not cover the binding site region or some regions, step,
(D) after (c) spreading the sample in a plate in a closed configuration, wherein the plates are facing each other and the spacer and associated volume of sample are located between the plates, There is a larger area of the sample in contact with the binding site than when the plate is in the open configuration, and the thickness of the relevant volume of the sample at the binding site is adjusted by the plate and spacer, and the relevant volume of the sample is A method comprising steps that are partial or total volume.

V2. A device for binding a target entity in a sample to a surface binding site, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a binding site on its surface that binds to a target entity in the sample, and the binding site when the sample is attached to only one plate and without contact with another plate, Has a larger area than the sample contact area,
One or two of the plates each include a spacer fixed to its corresponding plate and having a predetermined height,
One arrangement is that the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers, and that the sample adheres to one or two of said plates and at the time of attachment also the area of the binding site. It is an open structure that does not cover the area of the part,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates are opposed to each other, the spacer and the sample are located between the plates, and the plates are in the open configuration. , The sample is in contact with more areas of the binding site and the thickness of the sample on the binding site is adjusted by plates and spacers.

8-2 Addition of reagent to small volume sample by spreading sample V3. A method for binding a target entity in a sample to a binding site, the method comprising:
(A) a step of obtaining a first plate and a second plate that are movable with respect to each other so as to have different configurations, the first plate including a reagent added to a sample on its surface A storage site, one or two of said plates comprising spacers, and each spacer being fixed to its respective plate and having a predetermined height;
(B) depositing a sample on one or two of the plates when the plates are configured in an open configuration, wherein the two plates are partially or completely separated, The spacing between is not adjusted by spacers, and the sample does not contact some or some areas of the storage site during deposition, step,
(C) after (b), placing the plates in a closed configuration and spreading the sample, wherein the plates face each other and the spacer and associated volume of sample are located between the plates. As compared to when the plate is in the open configuration, the sample is in contact with more area of the storage site and the thickness of the relevant volume of the sample on the binding site is adjusted by the spacer so that the relevant volume is A method comprising the steps of being part of a sample located.

V4. A device for binding a target entity in a sample to a binding site, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a storage site on its surface containing a reagent to be added to the sample, one or two of the plates contains spacers, and each spacer is fixed on its respective plate and is predetermined. Has a height of
One or two of the plates each include a spacer fixed to its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is a closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other, the spacer and associated volume of sample are located between the plates, and the plates are open. Compared to when in the configuration, the sample is in contact with more areas of the storage site, the thickness of the relevant volume of the sample on the binding site is adjusted by the spacer, and the relevant volume of the sample is located at the storage site. A device that is part.

  In the method according to paragraphs V1 and V2 and the apparatus according to V3 and V4, in some cases, even if the sample adheres to the binding site region or the storage region due to the small amount of the sample being wettable on the surface, The contact area between the sample and the plate is smaller than the area of the binding site or storage site. Therefore, there is a need for widening, and particularly accurate spreading.

  The sample drop may be multiple drops, and in the closed configuration the drops coalesce into a film having a thickness less than the maximum thickness.

  In the present invention, in the method described in paragraphs V1 to V7 and the apparatus described in paragraphs V2 to V8, the volume of the sample attached to one plate or a plurality of plates (“sample volume”) is 0.001 pL (pico Liter), maximum 0.01 pL, maximum 0.1 pL, maximum 1 pL, maximum 10 pL, maximum 100 pL, maximum 1 nL (nanoliter), maximum 10 nL, maximum 100 nL, maximum 1 μL (microliter), maximum 10 μL, maximum 100 μL, maximum 1 mL (Ml), up to 10 mL, or a range between any two of these values.

9. Uniform binding or uniform addition (UAB) of reagents in areas with uniform sample thickness
For assays and chemistries, it is advantageous to make thin sample thickness uniform over a very large area. These examples include binding the entity of the sample to the surface binding sites, adding reagents into the sample, quantifying the relevant volume of the sample, quantifying the analyte, etc.

  Accuracy, uniformity, and ease of use are essential for methods of reducing and adjusting the thickness of a sample of related volume (partial or total volume) using two plates.

  One aspect of the present invention improves the accuracy, uniformity or ease of use of a method and/or apparatus for adjusting the thickness of a sample of related volume by compressing the sample with two plates. It is to be.

9.1 Method for uniformly binding the target entity in the sample into the binding site of the plate UAB1. A method for uniformly binding in the binding site of a plate, the method comprising:
(A) obtaining a sample containing a target entity capable of diffusing into the sample,
(B) obtaining a first plate and a second plate movable relative to each other in different configurations, the first plate being configured to bind to a target entity on its surface. One or two of the plates having bound binding sites, the spacers being immobilized on their corresponding plates and having a predetermined height,
(C) depositing the sample on one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) after (c) spreading the sample in a plate in a closed configuration, wherein the plates are facing each other and the spacer and associated volume of sample are located between the plates, The binding site contacts the relevant volume and the thickness of the relevant volume of the sample is adjusted by the plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration, and the binding site More uniform, including steps,
The spacer and plate are configured such that the adjusted thickness of the sample of the relevant volume in the closed configuration is more uniform than the adjusted thickness of the sample in the open configuration, the relevant volume being less than the volume of the sample. A method that is a portion or the entire volume.

  It also has storage sites on the corresponding plate of binding sites to form a uniform sandwich.

UAB2. A device for uniformly binding a target entity in a sample into a binding site of a plate, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a binding site on its surface configured to bind to a target entity, one or two of said plates each being fixed to its corresponding plate and having a predetermined height. Including a spacer having
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other and the volume of sample associated with the spacer is located between the plates and the binding site is When in contact with the relevant volume, the thickness of the relevant volume of the sample is adjusted by plates and spacers, when the plate is in the open configuration, the thickness is less than the maximum thickness of the sample and more uniform at the binding site And
The spacer and plate are configured such that the adjusted thickness of the sample of the relevant volume in the closed configuration is more uniform than the adjusted thickness of the sample in the open configuration, the relevant volume being less than the volume of the sample. A device that is a portion or the entire volume.

9.2 Method for uniformly adding reagent on plate into sample UAB3. A method for uniformly adding a reagent into a relevant volume of a sample, comprising:
(A) a step of obtaining a first plate and a second plate that are movable with respect to each other so as to have different configurations, the first plate including a reagent added to a sample on its surface Has a storage site, the reagent is soluble in the sample and can diffuse into the sample, one or two of the plates comprises spacers, and each spacer is fixed on its respective plate and has a predetermined height There are steps,
(B) obtaining a sample,
(C) depositing a sample on one or two of the plates when the plates are configured in an open configuration, the open configuration is such that the two plates are partially or completely separated and the spacing between the plates is Is not adjusted by the spacer, is a configuration, step,
(D) after (c) spreading the sample in a plate in a closed configuration, wherein the plates are facing each other and the spacer and associated volume of sample are located between the plates, The storage site is in contact with the relevant volume and the thickness of the relevant volume of the sample is adjusted by plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration. ,
The spacer and plate are configured to be more uniform than the thickness of the relevant volume of the sample, which is more uniform over the area of the relevant volume in the plate closed configuration than in the plate open configuration, and the associated volume Is a part of or the entire volume of the sample.

UAB4. A device for uniformly adding a reagent to a sample of a relevant volume, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a storage site for a reagent that is added to the volume of the sample associated with its surface, the reagent being capable of dissolving in the sample and diffusing into the sample, one or two of the plates. One includes a spacer, and each spacer is fixed to its corresponding plate and has a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other and the volume of sample associated with the spacer is located between the plates and the storage site is Contacting the volume of interest and adjusting the thickness of the volume of the volume of the sample by means of plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration,
The spacer and plate are configured to be more uniform than the thickness of the relevant volume of the sample, which is more uniform over the area of the relevant volume in the plate closed configuration than in the plate open configuration, and the associated volume Is the device, which is part of or the entire volume of the sample.

9.3 Method for uniformly forming capture-analyte-detection sandwiches on binding sites UAB5. A method for uniformly forming a capture-analyte-detection sandwich on a junction site of a plate, comprising: (a) obtaining a sample containing a target analyte;
(B) obtaining a capture agent and a detection agent that bind (capable) to the target analyte to form a capture agent-target analyte-detection agent sandwich;
(C) a step of obtaining a first plate and a second plate that are movable relative to each other so as to have different configurations, wherein the first plate has a binding site to which a capture agent is immobilized. And the second plate has a storage site for storing the detection agent, the reagent being capable of dissolving in the sample and diffusing into the sample when the storage site contacts the sample, and one or two of said plates A step of including spacers, each spacer being fixed to its corresponding plate and having a predetermined height;
(D) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and the plates are The spacing between is not adjusted by the spacer, is a step,
(E) after (d) spreading the sample in a plate in a closed configuration, wherein the plates face each other and the spacer and associated volume of sample are located between the plates, The thickness of the relevant volume of sample is adjusted by the plates and spacers, the thickness being less than the thickness of the sample when the plate is in the open configuration, the sample being in contact with the binding and storage sites,
The spacer and the plate are configured to be more uniform than the thickness of the relevant volume of the sample, which is more uniform over the area of the relevant volume in the plate closed configuration than in the plate open configuration. Is a part of or the entire volume of the sample.

UAB6. A device for uniformly forming a capture-analyte-detection sandwich on a plate junction site, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a binding site to which the capture agent is immobilized, which capture agent is capable of binding the target analyte in the sample,
The second plate (a) dissolves in the sample and diffuses into the sample when the storage site contacts the sample, (b) binds the target analyte and capture agent-target analyte-detector sandwich. Having a storage site for storing a detection agent that enables the formation of
One or two of the plates each include a spacer fixed to its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other and the volume of sample associated with the spacer is located between the plates The thickness of the sample of volume is adjusted by the plate and spacer, the thickness being less than the thickness of the sample when the plate is in the open configuration, the sample being in contact with the binding and storage sites,
The spacer and plate are configured to be more uniform than the thickness of the relevant volume of the sample, which is more uniform over the area of the relevant volume in the plate closed configuration than in the plate open configuration, and the associated volume Is the device, which is part of or the entire volume of the sample.

9.4 Uniform adjustment of the thickness of the relevant volume sample between the two plates UAB7. A method for adjusting the thickness of a sample of related volume, comprising:
(A) obtaining a sample, wherein the thickness of the sample of relevant volume is adjusted
(B) obtaining two plates that are movable relative to each other in different configurations, one or two of the plates having a predetermined spacer distance and height, A step including fixed spacers,
(C) depositing a sample on one or two of the plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) after (c) spreading the sample in a closed configuration with the plates in the closed configuration with the plates facing each other and the spacer and associated volume of sample located between the plates, The thickness of the sample of relevant volume is adjusted by the plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration;
The spacer and the plate are configured to be more uniform than the thickness of the relevant volume of the sample, which is more uniform over the area of the relevant volume in the plate closed configuration than in the plate open configuration. Is a part of or the entire volume of the sample.

UAB8. A device for adjusting the thickness of a sample of related volume, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The one or two plates include spacers having a predetermined distance and height between the spacers and fixed to the respective plates,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other and the volume of sample associated with the spacer is located between the plates and is associated. The thickness of the sample of volume is adjusted by plates and spacers, the thickness being less than the maximum thickness of the sample when the plate is in the open configuration,
The spacer and plate are configured to be more uniform than the thickness of the relevant volume of the sample, which is more uniform over the area of the relevant volume in the plate closed configuration than in the plate open configuration, and the associated volume Is the device, which is part of or the entire volume of the sample.

  In the method and apparatus of paragraphs U1-U8, the spacer and plate configurations that equalize the thickness of the relevant volume of the sample have embodiments described in this disclosure.

  Sample Thickness Uniformity In the method and apparatus of paragraphs U1 to U8, sample thickness uniformity of the relevant volume is that sample thickness in the closed configuration is 0.001% maximum and 0.01 maximum. %, max 0.05%, max 0.1%, max 0.5%, max 1%, max 2%, max 5%, max 10%, max 20%, max 30%, max 50%, max 75 %, up to 90%, less than 100%, or having a relative variation in range between any two of these values.

  In a preferred embodiment of the method and apparatus of paragraphs U1-U8, sample thickness uniformity of the relevant volume refers to sample thickness in the closed configuration of up to 0.1%, up to 0.5%, Having a relative variation of up to 1%, up to 2%, up to 5%, up to 10%, up to 20%, up to 30%, up to 50%, or a range between any two of these values. ..

  Another parameter that is very important for reducing saturation incubation time is sample thickness uniformity. With large variations in thickness across the binding site, the saturation incubation time depends on the position of the binding site, forcing a longer saturation incubation time and ensuring that all positions in the binding site reach saturation.

10. Amplified surface One of the traditional major obstacles to either assay using PoC diagnostics and small sample volumes is low sensitivity. It is desirable to enhance the signal of the assay. One aspect of the present invention relates to devices and methods that achieve high sensitivity by placing binding sites on a signal amplification surface (SAS) and amplifying the signal. The signal amplification surface is also called the signal amplification layer (SAL).

  The general structure of SALs includes nanoscale metal-dielectric/semiconductor-metal structures that amplify local surface electric fields and gradients and optical signals. Amplification is high between locations with sharp (ie, high curvature) edges of the metal structure and small gaps between the two metal structures. The highest amplification area is the area with sharp edges and small gaps. Moreover, the dimensions of all metallic and non-metallic micro/nanostructures are typically smaller than the wavelength of light (ie, subwavelength) that SAL amplifies.

  In some embodiments, the SAL layer has as many metal sharp edges and small gaps as possible. This requires having closely packed groups of metallic nanostructures with small gaps between the nanostructures. The SAL structure may include several different layers. Further, the SAL layer itself is filed on March 15, 2013, US Provisional Application No. 61/801,424, filed March 15, 2014, which is hereby incorporated by reference for various purposes. PCT Application WO2014197096, as well as PCT/US2014/028417 (Chou et al., "Analyte Detection By Targeted Immunization Reconciliation Affiliation Reconciliation, Inc.") for various purposes. Further improvements can be made by a process that can further cover portions of metal material without sharp edges and small gaps, as described.

  One particular embodiment of a signal amplification surface comprises at least a portion at least a portion of the metal dot structure, the metal disk and/or the metal backplane, and optionally a capture agent that specifically binds to the analyte. A D2PA array (disk-coupled dot-on-pillar antenna array) containing a covering molecular adhesion layer, the capture agent binding to the D2PA array. When the analyte binds to the capture agent, the nanosensor can amplify the light signal from the analyte. In some embodiments, the dimensions of one, some or all of the critical metal and dielectric components of the SAL are smaller than the wavelength of light being detected. Details of the physical structure of disk-coupled dot-on-pillar antenna arrays, methods of making them, methods of coupling capture agents to disk-coupled-dot-on-pillar antenna arrays, and disk couples Methods for detecting analytes using do-dot-on-pillar antenna arrays are described in WO2012024006, WO2013154770, Li et al. (Optics Express 2011 19, 3925-, incorporated herein by reference for various purposes. 3936), Zhang et al. (Nanotechnology 2012 23:225-301), Zhou et al. (Anal. Chen. 2014 84:4489).

10.1 Amplification of signal assaying target entity in relevant volume of sample M1. A method for amplifying a signal for assaying a target entity in a sample of relevant volume, comprising:
(A) obtaining a sample containing the target entity,
(B) obtaining two plates that are movable relative to each other in a different configuration, one plate being attached to its surface for binding to a target entity and on or near the signal amplification surface. One binding site comprising a signal amplification surface configured to amplify an optical signal, one or two of said plates comprising spacers, each spacer being on its corresponding plate and predetermined. Having a height of
(C) depositing a sample on one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are separated and the spacing between the plates is a spacer. Not adjusted by, composition, step,
(D) after (c) spreading the sample in a plate in a closed configuration, wherein the plates are facing each other and the spacer and associated volume of sample are located between the plates, Adjusting the thickness of the relevant volume of the sample by the plate and the spacer, the spacer being thinner than the spacer when the plate is in the open configuration, the relevant volume of the sample contacting the binding site; and (e ) After (e), incubate for a period of time while the plate is in the closed configuration to allow the target entity in the relevant volume of the sample to bind to the binding site. A step,
The method, wherein the volume of interest is the portion where the sample contacts the binding site when the plate is in the closed configuration.

M2. A device for amplifying a signal for assaying a target entity in a sample of relevant volume, comprising:
A first plate and a second plate movable relative to each other in different configurations,
The first plate has on its surface a signal amplification surface configured (i) to bind to a target entity in the sample and (ii) to amplify an optical signal on or near the signal amplification surface. Including one binding site,
One or two of the plates each include a spacer located on its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the sample is deposited in the open configuration, in which the plates face each other and the volume of sample associated with the spacer is located between the plates and is associated. The volume sample thickness is adjusted by the plates and spacers, which is less than the thickness when the plate is in the open configuration,
The volume of interest is the device where the sample contacts the binding site when the plate is in the closed configuration.

  In some embodiments, the signal-amplifying surface comprises at least one of metal-dielectric nanostructures, metal-semiconductor nanostructures, and disk coupled dot-on-pillar antenna arrays.

  In some embodiments, the signal amplification surface comprises a metal layer.

11 Reagent volume savings in rapid binding during assay (S)
If the entity in the reagent binds to the binding site on the surface (eg coating the plate with a capture agent or stains the biological sample surface), it is desirable to have a short incubation time. One way to obtain a short incubation time is to significantly increase the concentration of entity in the reagent. However, such a method allows only a small portion of the entity in the reagent near the binding site to reach and bind to the binding site and the rest too far from the entity to diffuse and bind to the binding site in a short incubation time. Instead, it is useless and wasteful, which wastes the entity and thus costs. For typical diffusion constants of common reagents in common solutions, typical diffusion lengths are about 10 μm, 33 μm, and 100 μm for incubation times of 1 s (sec), 10 s, and 100 s, respectively. . A typical thickness of a droplet on a typical surface is 2.5 mm, which is at least 25 times thicker than the above diffusion length, and a significant waste (cost) if the incubation time is 100 s or less. Bring One aspect of the present invention is to spread the droplets of reagent very thin over a large area (thinner than the natural drop), saving reagent and reducing cost.

11-1 A method for saving a reagent containing a target entity that binds to a surface binding site in a reagent by spreading the reagent (having a binding site whose volume is smaller than a natural contact region)
S1. A method for conserving a reagent containing a target entity that binds to a surface binding site, the method comprising:
(A) Obtaining a first plate and a second plate that are movable relative to each other in different configurations, the first plate having a binding site on its surface, One or two comprising spacers each fixed to its corresponding plate and having a predetermined height;
(B) a step of obtaining a reagent, wherein the reagent comprises (i) a target entity capable of binding to a binding site, and (ii) a contact region of the reagent attached to the binding site without contact with another plate. Has volume and wetting properties such that is smaller than the area of the binding site,
(C) depositing the sample on one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) After (c), the plate is in a closed configuration and the sample is unfolded, wherein the plates face each other, the spacer and the sample are located between the plates, and the plate is in an open configuration. There is a larger area of the sample in contact with the binding site than when it is in, and the thickness of the sample at the binding site is adjusted by the plates and spacers, the thickness being less than the thickness when the plate is in the open configuration. Including the method.

  The method of paragraph S1, which further comprises after (d) incubating the sample for a period of time and stopping the incubation while the plate is in the closed configuration, the incubation time being the plate in the closed configuration. At some point the target entity is approximately equal in time to diffuse through the maximum sample thickness, and incubation is the process that allows the entity to bind to the binding site.

S2. A device for conserving a reagent containing a target entity that binds to a surface binding site,
A first plate and a second plate movable relative to each other in different configurations,
The first plate has a binding site on its surface that binds to the target entity in the reagent, and the binding site when the reagent is attached to only one plate without contact with another plate is Has a large area,
One or two of the plates each include a spacer fixed to its corresponding plate and having a predetermined height,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of said plates,
Another configuration is the closed configuration, which is configured after the reagents are deposited in the open configuration, where the plates are opposite each other, the spacer and the reagent are located between the plates, and the plates are in the open configuration as compared to when in the open configuration. The device wherein the sample contacts more area of the binding site and the thickness of the reagent on the binding site is adjusted by the plate and spacer, the thickness being less than the thickness when the plate is in the open configuration.

12. Volume and/or concentration detection and/or quantification (Q)
Quantifying and/or controlling the relevant volume of a sample is useful for quantifying and/or controlling the concentration of chemical compounds (including analytes, entities, reagents, etc.) in a sample.

  Common methods for quantification of sample volume include the use of metering pipettes (eg Eppendorf's "Research Plus Pipette, Adjustable, 0.5-10 μL", SKU #3120000020), or geometry. For PoC (point of care) or household use, such metering devices are difficult and/or expensive to use. There is a need for simpler and cheaper methods and devices for quantifying and/or controlling sample volume and/or concentration.

  One aspect of the invention relates to methods, devices, and systems for quantifying and/or controlling the relevant volume of sample deposited on a plate without the use of metering pipettes and/or fixed microfluidic channels. The relevant volume, which may be part or the whole of the sample volume, relates to quantifying and/or controlling the concentration of the target analyte and/or entity in the sample. The method, apparatus and system of the present invention are easy to use and low cost.

12.1 Methods for Quantifying Relevant Volumes of Samples Q1. A method for quantifying the relevant volume of a sample, comprising:
(A) obtaining a sample in which the relevant volume of the sample is quantified,
(B) Obtaining two plates that are movable relative to each other in different configurations, wherein one or two of the plates have a predetermined spacer distance and height, respectively. Step, including a spacer fixed to the plate of
(C) depositing the sample on one or two of the two plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and A configuration in which the spacing between the plates is not adjusted by spacers; and (d)(c), after which the plates are placed in a closed configuration and the sample is spread, the plates facing each other in the closed configuration. And the spacer and associated volume of sample are located between the plates, and the thickness of the associated volume of sample is adjusted by the plate and the spacer, the thickness being the maximum of the sample when the plate is in the open configuration. Thinner than the thickness of the at least one spacer located in the sample,
(E) quantifying a relevant volume of the sample while the plate is in the closed configuration, the relevant volume being at least a portion of the total volume of the sample. .

Q2. In some embodiments, the method for quantifying the relevant volume of a sample comprises:
(A) obtaining a first plate and a second plate,
(B) producing a sample to be quantified between two plates,
(C) deforming the shape of the sample by compressing the two plates to reduce the thickness of the sample,
(D) step of laterally spreading the sample between the plates,
Quantifying a relevant volume of the sample while the plate is in the closed configuration, the relevant volume being at least a portion of the total volume of the sample.

12.2 Plates for quantifying relevant volumes in samples Q3. A plate for quantifying the relevant volume in a sample, comprising:
A sample for contacting on its surface (i) a spacer having a predetermined distance and height between the spacers and fixed to the surface, and (ii) a sample having an associated volume quantified with the sample. A contact area and the at least one spacer comprises a plate within the sample contact area.

12.3 Device for quantifying relevant volumes in a sample Q4. A device for quantifying a relevant volume in a sample, comprising:
Includes a first plate and a second plate, which are (a) movable relative to each other in different configurations, and (b) each in contact with a sample having an associated volume to be quantified. Has a sample contact area for
One or two plates include spacers on their surface, said spacers having a predetermined distance and height between the spacers, fixed to each plate,
One configuration is an open configuration in which the two plates are separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates,
Another configuration is the closed configuration, where the samples are configured after being deposited in the open configuration, in which the plates face each other and the spacers and associated volume of sample are located between the plates and the associated The thickness of a volume of sample is adjusted by the plate and spacer and is smaller than when the plate is in the open configuration, the at least one spacer being inside the sample,
The apparatus, wherein the relevant volume of the sample is quantified in the closed configuration and the relevant volume is at least a portion of the total volume of the sample.

12-5. Measurement of the relevant volume of the sample MS1. In the present invention, quantification of the relevant volume of the sample while the plate is in the closed configuration includes, but is not limited to, each of the following five embodiments:
(A) measuring the relevant volume of the sample by mechanical, optical, electrical, or any combination thereof methods;
(B) independently measuring one or more parameters associated with the relevant volume of the sample using a method selected from mechanical, optical, electrical, or any combination thereof.
(C) using one or more predetermined parameters related to the relevant volume of the sample (ie the sample parameters determined before the plate was in the closed configuration);
(D) (i) measuring one or more parameters associated with the volume of interest when the plate is in the closed configuration; Determining the relevant volume of the sample by predetermining the parameters of
(E) determining the non-sample volume,
(F) Any combination of the above (ie, a, b and c).

  In the method of paragraph MS1, the mechanical method is a spacer (ie, a mechanical device that adjusts the spacing between the inner surface of the substrate and the cover plate to a predetermined value), a mechanical probe or ruler, a sound wave (eg, to measure the spacing). Ultrasonic reflection and/or interference), or any combination thereof.

  The method of paragraph MS1, wherein optical interference, optical imaging (eg, taking a 2D (2D)/3D (3D) image of the sample), multiple optical imaging (different viewing angles, different wavelengths, different phases and/or (With different polarizations), image processing, or the use of any combination thereof, but not limited thereto.

  Electrical methods include, but are not limited to, measuring capacitance, resistance, or impedance, or any combination thereof.

  In the method of paragraph MS1, in some embodiments, measuring the sample thickness is measuring the distance between the inner surfaces of the two plates.

  The method of paragraph MS1, in some embodiments, employs a sample of a predetermined one or more parameters related to the relevant volume of the sample, the predetermined parameter being adjusted by the spacer when the plate is in the closed configuration. It is the predetermined sample thickness which was made.

  In the method of paragraph MS1, in some embodiments, a sample of a predetermined one or more parameters related to the relevant volume of the sample is used, the predetermined parameter is a predetermined spacer height.

  In the method of paragraph MS1, in some embodiments, the relevant volume-related parameter of the sample is the parameter in the closed configuration, which is (i) (in CROF) the inner surface of the first plate and The distance between the inner surfaces of the two plates, (ii) sample thickness, (iii) the entire sample area or relevant parts, (iv) the entire sample volume or related parts, or (v) those Including, but not limited to, any combination.

  In the method of paragraph MS1, in some embodiments, quantifying the sample volume or related sample volumes comprises: (i) multiplying the sample thickness by the total sample area to obtain the total sample volume; (ii) ) Multiplying the sample thickness with the relevant sample area to obtain the relevant sample volume, or (iii) multiplying the relevant sample thickness with the total or relevant sample area to obtain the relevant sample The step of obtaining a volume is included.

  In the method of paragraph MS1, in some embodiments the measurement is taking a 3D (three-dimensional) image of the volume of interest.

  In the method of paragraph MS1, in some embodiments, quantifying the relevant volume of the sample measures the side area of the relevant volume of the sample and then uses the thickness of the relevant volume. And the thickness of the relevant volume is determined from the spacer information, the spacer information including the spacer height.

  In the method of paragraph MS1, in some embodiments, quantifying the relevant volume of the sample includes measuring the side area of the relevant volume of the sample and the spacer together, and then measuring the thickness of the relevant volume. And the volume of the spacer are used to determine the volume of the sample of the relevant volume, and the thickness of the relevant volume is determined from the spacer information.

  In the method of paragraph MS1, in some embodiments, quantifying the relevant volume of the sample is performed by measuring the side area and thickness of the relevant volume of the sample.

  In the method of paragraph MS1, in some embodiments, quantifying the relevant volume of the sample is performed by optically measuring the volume of the sample of the relevant volume.

  In the method of paragraph MS1, in some embodiments scale marks are used to help quantify the volume of the relevant sample while the plate is in the closed configuration, and some embodiments of scale markers. , Their use, measurement, etc. are described in Section 2.

  In the method of paragraph MS1, in some embodiments, quantifying the relevant volume of the sample comprises subtracting the non-sample volume, and in some embodiments, the non-sample volume is described in the present invention. Determined by the embodiment.

12-4. Methods for Quantifying Analyte Concentration in Relevant Volumes of Samples Q5. A method for quantifying an analyte in a sample of relevant volume, comprising:
(A) performing the steps in the method of paragraph Q1, and (b) measuring the signal associated with the analyte from the relevant volume after step (a), wherein the relevant volume is The method comprises the step of being at least a portion of the total volume of the sample.

Q6. A method for quantifying an analyte in a sample of relevant volume, comprising:
(A) performing the steps in the method of paragraph Q2, and (b) measuring the signal associated with the analyte from the relevant volume after step (a), wherein the relevant volume is The method comprises the step of being at least a portion of the total volume of the sample.

  The method of any of paragraphs Q5-6, in some embodiments, further comprising splitting the signal associated with the analyte from the sample of the relevant volume based on the volume of the relevant volume, The step of calculating the analyte concentration is included.

  In the method of any of paragraphs Q5-6, the one or two plates further comprises a binding site, a storage site, or both.

  In the method of any of paragraphs Q5-6, in some embodiments, the signal associated with the analyte is a signal directly from the analyte or from a label attached to the analyte.

Q7. A method for quantifying an analyte in a sample of relevant volume, comprising:
(A) performing one or more of the steps in the method of paragraph Q1 characterized in that one or two plates contain binding sites; and (b) after step (a), the analyte from the relevant volume is Measuring the relevant signal, said relevant volume comprising the step of being at least a portion of the total volume of the sample.

Q8. A method for quantifying an analyte in a sample of relevant volume, comprising:
(A) performing one or more of the steps in the method of paragraph Q2, characterized in that one or two plates contain binding sites; and (b) after step (a), the analyte from the relevant volume is A method comprising measuring a relevant signal, said relevant volume being at least a portion of a total volume of the sample.

  In the method of any of paragraphs Q7-8, in some embodiments, the signal associated with the analyte is attached to the signal from the analyte directly bound to the binding site or to the analyte bound to the binding site. The signal from the sign.

12.5 Plates for Use in Quantifying Analyte Concentrations in Relevant Volumes in Samples Q9. A plate for use in quantifying an analyte concentration in a relevant volume in a sample, comprising:
On its surface, including (i) spacers having a predetermined distance and height between spacers, and (ii) a sample contact area for contacting a sample having an analyte concentration in the relevant volume to be quantified. , At least one spacer comprises a plate within the sample contact area.

12.6 Apparatus for use in quantifying analyte concentration in a relevant volume in a sample The number of target analytes and/or entities in a sample is quantified and also the relevant volume of the sample Once quantified, the concentration of target analyte and/or entity in the sample can be quantified or controlled.

Q10. A device for quantifying an analyte concentration in a relevant volume in a sample, comprising:
Including a first plate and a second plate, the first plate and the second plate are (a) movable relative to each other in different configurations, and (b) each is quantified. Having a sample contacting area for contacting a sample having an analyte concentration in a volume of interest, one or two plates comprising spacers on their surface having a distance and height between the predetermined spacers, Each spacer is fixed to its plate,
One configuration is an open configuration in which the two plates are separated and the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates,
Another configuration is the closed configuration, where the samples are configured after being deposited in the open configuration, in which the plates face each other and the spacers and associated volume of sample are located between the plates and the associated The thickness of a volume of sample is adjusted by the plate and spacer and is smaller than when the plate is in the open configuration, at least one spacer is inside the sample and the analyte concentration in the relevant volume of the sample. The apparatus is quantified in a closed configuration and the relevant volume is at least a portion of the total sample volume.

  In the device of any of paragraphs Q9 and Q10, the plate further comprises a binding site or a storage site or both. One embodiment of the binding site is the binding site bound to the analyte in the sample.

  In both the devices of paragraphs Q9 and Q10, the scale mark, the plate further comprises one or more scale markers, and some embodiments of the scale markers are described in Section 2.

  In the methods or devices of any of paragraphs Q1-10, in some embodiments the metrology device comprises at least one of an imaging device and a camera.

  In the method or apparatus of any of paragraphs Q1-10, in some embodiments, the metrology device is configured to image a lateral region of the sample of relevant volume.

  In the method or apparatus of any of paragraphs Q1-10, in some embodiments, the metrology device includes a light source for illuminating a lateral region of the sample of relevant volume.

  In the method or apparatus of any of paragraphs Q1-10, in some embodiments, the step of calculating the concentration is dividing the total target analyte or entity by the relevant sample volume.

  In the method or apparatus of any of paragraphs Q1-10, in some embodiments, the metrology signal uses an optical imaging device to count the target analyte or entity. For example, the measurement may be measurement of blood cells (red blood cells, white blood cells, platelets) in the blood sample using an optical microscope.

  In the method or apparatus of any of paragraphs Q1-10, in some embodiments, measuring the number of target analytes or entities in the sample is performed by performing a surface-immobilized assay that captures the target analytes or entities on the surface. It may be in the form.

  An apparatus for quantifying the volume of a sample or detecting/quantifying an analyte in a sample is the apparatus of any of paragraphs Q1-10, (1) an optical imaging device, and/or (2) a light source and It includes an optical imaging device and the like. Optical imaging devices include photosensors, optical lenses, filters, polarizers, wave plates, beam splitters, mechanical mounts, or any combination thereof.

  In some embodiments, the relevant sample area or volume measurements include (i) having a marker on the first plate, the cover plate, between them, or any combination thereof, (ii) optical imaging (E.g., capturing a 2D (2D)/3D (3D) image of the sample), performing the imaging multiple times with different viewing angles, different wavelengths, different phases, and/or different polarizations, and ( iii) processing the image based on the manufacturer and sample images. Relevant means related to the determination of target analyte concentration.

  Scanning In some embodiments, reading the signal from the sample uses a scanning method, where a reader (eg, a photodetector or camera) reads a portion of the sample (or plate) and then Moving to another part of the sample (or plate), such a process is carried out until a particular predesignated part of the sample (or plate) is read. Scanning and reading on the sample covers all parts of the sample (or plate) or part of the sample (or plate). In some embodiments, scanning and reading are aided by position markers that indicate the sample (or plate). An example of a position marker is a periodic spacer with a fixed period and position, or a marker for an associated area that also has a predetermined position and size that indicates the position of the sample or plate.

13 Detection and quantification of analytes and others (D)
In certain embodiments, detecting and/or quantifying (ie, assaying) an analyte by measuring a signal associated with the analyte, which signal is an optical signal, an electrical signal, a mechanical signal, a chemo-physical signal. Target signal, or any combination thereof. In some embodiments, the analyte assay is performed when the two plates in the CROF device are close together. In some embodiments, the analyte assay is performed when the two plates in the CROF device are separated from each other.

  The optical signal includes light reflection, scattering, transmission, absorption, spectrum, color, luminescence, intensity, wavelength, position, polarization, luminescence, fluorescence, electroluminescence, chemiluminescence, electrochemiluminescence, or any combination thereof. , But not limited to them. An optical signal is in the form of an optical image (ie, optical signal and sample or device position) or the total number of all photons from a given area or volume. The preferred wavelength of light is in the range of 400 nm to 1100 nm, in the range of 50 nm to 400 nm, in the range of 1 nm to 50 nm, or in the range of 1100 nm to 30000 nm. Another preferred wavelength is terahertz.

  The electrical signal includes, but is not limited to, charge, current, impedance, capacitance, resistance, or any combination thereof. Mechanical signals include, but are not limited to, mechanical waves, sound waves, shock waves, or vibrations. Chemi-physical signals include, but are not limited to, PH values, ions, heat, bubbles, color changes produced in the reaction.

  For example, the label is a bead, and the label is an analyte-specific binding process (ie, binding a bead to an analyte with a detection agent, capturing an analyte and beads with a capture agent, capturing An agent is used to bind the analyte and then a bead with the detection agent to attach the label, or by other means) to the label. Subsequent counting is used to identify each bead attached to the analyte and count them.

  In some embodiments, each analyte or bead is labeled with optical means (eg, (i) optical label and label reading, (ii) surface plasmon resonance, (iii) optical interference, (iv) electrical methods (eg, Sensing and counting by means of capacitance, resistance, impedance, etc.) or other means, the sensor can be located on the surface of the first plate and/or the second plate.

  Certain embodiments include determining the analyte concentration in (a) surface immobilization assay, (b) bulk assay (eg, hemacytometer) and (c) others. In some embodiments, the method for sample volume, relevant volume of sample or concentration uses a smartphone.

  In the methods or apparatus of any of paragraphs Q1-10, in some embodiments, the signal measurement measures the number of analytes in the sample or the number of labels attached to the analytes in the sample. That is. In another embodiment of paragraph Q5, the "measurement signal" identifies (a) each analyte or each label attached to each analyte, and (b) counts them.

  In some embodiments, analyte detection is achieved when electrodes are placed on one or both of the first and second plates, which applies to any method and apparatus that uses CROF. , Electric way. The electrodes measure the charge, current, capacitance, impedance or resistance of the sample, or any combination thereof. The electrodes measure the electrolyte in the sample. The electrode has a thickness equal to or less than the thickness of the spacer. In some embodiments, the electrodes function as part of the spacer. The electrodes are made of various electrically conductive materials. Preferred electrode materials are gold, silver, aluminum, copper, platinum, carbon nanotubes, or any combination thereof.

  In the method or apparatus of any of paragraphs Q1-10, in some embodiments the metrology uses a device that is a camera or a photodetector and a processor configured to perform the metrology.

  In the methods or apparatus of any of paragraphs Q1-10, in some embodiments, the concentration determination device is configured to determine concentration based on measurements (volume, area, thickness, number of analytes, intensity). Included processor.

  The method or apparatus of any of paragraphs Q1-10, in some embodiments, determining the concentration of the target analyte in the relevant volume based on the measured side area, thickness, and number of target molecules. The apparatus further includes a concentration determination device configured to

Details of Signal Detection Using Pixelated Reading and Analysis The present invention, in some embodiments, detects signals from a sample, analyte, entity, binding site, reagent, CROF plate, or any combination thereof. And analyze. Some embodiments of signal detection using pixelated reading and analysis are described in the present invention, and some other embodiments are disclosed in Publication No. WO2014144133A and Application No. PCT/US2014/028417 (Chou et al., “Analyte. Detection Enhancement By Targeted Immobilization, Surface Amplification, And Pixelated Reading And Analysis"), which are incorporated herein by reference for all purposes.

  In some embodiments, the signals are electromagnetic signals including electrical and optical signals having different frequencies, light intensities, fluorescence, chromaticities, luminescence (electrical and chemiluminescent), Raman scattering, time resolved signals (including blinking). Is. The signal may also be a force due to local electrical, local mechanical, local biology or local optical interaction between the plate and the reader. The signal further includes the spatial (ie location), temporal and spectral distribution of the signal. The detection signal can also be absorbed.

  Analytes include proteins, peptides, DNA, RNA, nucleic acids, small molecules, cells, and nanoparticles of different shapes. The target analyte may be in solution or air or gas phase. Detection includes detection of presence, quantification of concentration, determination of target analyte status.

  In some embodiments, electric fields are used to aid molecular selectivity, or binding and detection.

Detection/Reading Methods In some embodiments of optical detection (ie, detection by electromagnetic radiation), the methods include far-field optical methods, near-field optical methods, epifluorescence spectroscopy, confocal microscopy, two-photon microscopy, And, but not limited to, total internal reflection microscopy, target analytes are labeled with electromagnetic radiation emitters and the signals in these microscopes can be amplified by the amplification surface of CROF plates.

  In some embodiments, the signal includes information about position, local strength, local spectrum, local polarization, local phase, local Raman signature of the signal, or any combination thereof.

  In some embodiments, signal detection is the measurement of a collective signal from an area (ie, the signal from the area regardless of its position within the area).

  In certain embodiments, signal detection is the detection of a signal image (i.e., signal and position) of an area, i.e., dividing the area into a plurality of pixels and detecting the signal in each pixel of the area. It is to detect individually and is also called “PIX” or “pixelated image detection”. The individual measurements of each pixel may be parallel or sequential or mixed.

  In some embodiments, the reading detects the signals sequentially or in parallel or a combination thereof using a suitable detection system. In sequential detection, one or more pixels are detected at once, and a scanner is used to move the detection to other areas of the SAL. In parallel detection, a multi-pixel detector array such as an imaging camera (eg CCD camera) is used to detect signals from different pixels simultaneously. Scanning is a single pass or multi-pass with different pixel sizes. FIG. 2C of PCT/US2014/028417 schematically shows a pixelated reading in the x, y, z states.

  The pixel size for read/detect is adjusted for a balance of optical resolution and total read time. Smaller pixel sizes take longer to read/scan all or part of the SAL. A general pixel size is 1 μm to 10 μm in size. The pixel shape is a circle, a square, or a rectangle. The lower limit of pixel size is determined by the optical resolution of the microscope system, and the upper limit of pixel size is determined to avoid reading errors due to non-uniform optical response of the imager (optical aberration, illumination uniformity, etc.). .

Reading System Referring to the drawings of PCT/US2014/028417, one embodiment of the reading system represents (a) one or more plates for CROF and (b) individual target analyte binding events. A reader 205 for generating an image of the signal emanating from the surface of the plate, (c) a device assembly 300 for holding the plate and the imager, and (d) electronics and data storage for storing the image. 301, and (e) a computer containing programming for identifying and counting individual binding events within an area of the image.

  The instrument assembly 300 controls and modifies the relative position between the plate and the reader in at least one of the three (x, y, z) orthogonal directions to read the signal. An embodiment of the device assembly includes a scanner 301. In some embodiments, the scanner 301 scans in at least one of the three (x,y,z) orthogonal directions.

  In some embodiments, reader 302 is a CCD camera. In some embodiments, the reader 302 includes one or more other optics selected from optical filters 303, spectrometers, lenses 304, apertures, beam splitters 305, mirrors 306, polarizers 307, wave plates and shutters. It is a photodetector including a device. In some embodiments, the reading device 302 is a smartphone or cell phone with local and remote communication capabilities. The reader collects the position of the signal, the local intensity, the local spectrum, the local Raman signature, or any combination thereof.

  In some embodiments, optical filter 303, light beam splitter 305, optical fiber, photodetector (eg, pn junction, diode, PMT (photomultiplier tube), or APD (avalanche photodiode), imaging camera (eg, , CCD camera or mobile phone camera)), and a spectrometer, together with a scanner provided by the device assembly 301, are coupled to the microscope system using a far-field confocal or wide-field setting.

  In some embodiments, in the confocal setting, the reading is performed by recording the brightness, the temporal and spectral changes of one or more pixels once, and raster scanning the entire area of interest of the SAL. In some embodiments, in the wide field of view setting, the camera is used to record the brightness and time variations of all or part of the SAL area. In some embodiments, suitable optical filters and light beam manipulators (polarizers, beam splitters, optical fibers, etc.) are required to ensure that only the desired signal is collected and detected. .. FIG. 9 of PCT/US2014/028417 schematically shows one arrangement of components for this system.

  Pixelated Analysis (PIX) In some embodiments of PIX, the signal detected in a pixelated manner is used to determine the number and/or type of a particular molecule at a particular pixel or pixels. Analyzed, which in turn is used to quantify the type and/or concentration of the target analyte. The term “signal detection by a pixelation method” refers to a method of dividing a region having a signal into a plurality of pixels and individually detecting a signal from each pixel of the region, and is “PIX” or “pixelated image”. Also called “detection”. The individual measurements of each pixel may be parallel or sequential or mixed.

  In some embodiments, the analyzing comprises analyzing spatial, tempo, spectral information of the signal. In some embodiments, analysis includes, but is not limited to, statistical analysis, comparison, consolidation, etc. FIG. 5 of PCT/US2014/028417 shows a flow chart of one embodiment of the method.

  Analysis-1 Method Analysis-1 of one embodiment of signal analysis includes (1) determining the local background signal strength, (2) determining the local signal strength for one marker, two markers, etc. And (3) determining the total number of markers in the imaging area.

  A background signal is a signal generated under the same conditions as other samples, except when the sample does not contain any target analyte.

  One embodiment of Analysis-1 uses an EM-CCD to record spatially distributed bioassay signal intensities. In other implementations, mobile phones (smartphones, mobile phones) are used to image the signal.

  Some details of Analysis-1 are as follows.

(1) Determine the strength of the local background signal. A reference sample is used to determine the background signal. This reference sample is a plate without any immobilized analyte and is imaged using the same instrument set under the same experimental conditions for bioassay on the plate. Then the intensities of all pixels in the image are plotted in a histogram giving the number of pixels at a particular signal intensity. Then, the signal strength having the most corresponding number of pixels is determined as the background signal background. This background intensity, along with their standard deviation (s.d.), is used to determine the threshold defined to distinguish between local background and local hotspot, where threshold=background+n ^* s. ． It becomes d. Here, n is an integer used as a parameter for adjusting the threshold value. Generally, in this work n is set to 3, 5 or 7.

(2) For a single bright pixel (I _x,y >threshold), the local signal strength of the beacon is determined using a two step procedure. First, time-evolving imaging of the sample is used to find hot spots with a single label (analyte). The total imaging time is on the 10 second scale and the resolution is on the 10 millisecond scale. For a single analyte hotspot, a clear ON/OFF binary behavior of hotspot fluorescence intensity is observed. Pixels displaying such activity are initially counted as a single label/analyte. Therefore, their coordinates and intensities on the image are recorded. The average intensity of these hotpots is then used as the intensity of a single label on the plate assay.

Secondly, bright pixels that do not show such binary behavior therefore show multiple labels/analytes. The signal strengths are then compared to the average brightness of a single sign to count the number of signs in the local hotspot. Alternatively, another simplified method based on the principle of Poisson statistics is used. At low concentrations of analyte (<1 pM), we observed a Poisson distribution with the probability that a small amount of analyte was immobilized at a high density of plasmon hot spots (approximately 2.5×10 ⁷ mm ⁻² ), which is two It means that there is a low probability that more than one analyte will be located in the same plasmon hotspot. For example, for a 1 fM target analyte, the probability that more than two labels will be located within the imaged region is less than 0.01% (estimated). Therefore, all bright hotspots that do not show single label behavior contain only two labels.

  (3) After completing (1) and (2), a list of hotspot pixel coordinates, intensities and corresponding label numbers can be tabulated. The total number of labels is obtained by SUM for each bright pixel label number.

  Analysis-2 Method Analysis-2 of one embodiment of signal analysis includes (1) determining a local background signal spectrum, (2) a local signal spectrum, such as one label, two labels. Determining, and (3) determining the total number of markers in the imaged area.

  Analysis-2 is based on recording the spatial distribution of the bioassay signal spectrum using a high resolution spectrometer in combination with a confocal microscope device. Some details of Analysis-2 are as follows.

(1) Use a reference sample to determine the background signal. This reference sample is the detector plate without any immobilized analyte and is imaged using the same instrument set under the same experimental conditions for the bioassay on the detector plate. The confocal microscope is then used to measure the local bioassay signal spectrum. The detection area is determined by the pinhole size in front of the high resolution spectrometer and the numerical aperture of the microscope objective. The confocal microscope raster scans over the sensing site of the sensing plate to obtain the spatial distribution of the background signal spectrum I(x,y,λ). Next, a histogram is plotted that gives the number of pixels with a particular spectral moment (∫I(λ)dλ). Similar to analysis-1 step (1), the spectral moment with the largest pixel is used as the background signal and its standard deviation is used to determine the threshold. I(λ) _threshold =I(λ) _background +n ^* s. d(λ). Here, n is an integer used as a parameter for adjusting the threshold value. Generally, in this work n is set to 3, 5 or 7. (2) Collect the spectrum of a single bright pixel using a confocal microscope device coupled to a high resolution spectrometer. Reading is performed in the same manner as in step (1). Single molecule spectra can be reliably detected using only sensitive CCDs with exposure times of a few seconds, so they cannot provide sufficient time resolution to determine the binary behavior of single labels at hotspots. .. Therefore, the spectral moments between different bright pixels are compared to determine the number of landmarks at the bright pixels. Due to the large expansion of the detection plate, single or multiple labels can be distinguished from the background. Therefore, the quantity of analytes in the hotspot can be determined.

  (3) After completing (1) and (2), a list of hotspot pixel coordinates, spectral moments and corresponding label numbers can be tabulated. The total number of labels is obtained by SUM for each bright pixel label number.

  Analysis-3 (Detected by Pixelated SERS Signal) Analysis-3 of one embodiment of signal analysis determines (1) the local background signal of the "Surface Enhanced Raman Scattering" (SERS) signature. And (2) determining the local SERS of one marker, two markers, etc., and (3) determining the total number of markers in the imaged region.

  Analysis-3 is based on recording the spatial distribution of the bioassay signal SERS spectrum using a high resolution spectrometer in combination with a confocal microscope device. Some details of Analysis-3 are as follows.

(1) Use a reference sample to determine the background signal. This reference sample is the detector plate without any immobilized analyte and is imaged using the same instrument set under the same experimental conditions for the bioassay on the detector plate. The local bioassay SERS spectrum is then measured using a confocal microscope. The detection area is determined by the pinhole size in front of the high resolution spectrometer and the numerical aperture of the microscope objective. The confocal microscope raster scans the entire sensing site of the sensing plate to obtain the spatial distribution of the background signal spectrum I(x,y,cm ⁻¹ ). For a particular biomolecule, a histogram is constructed giving the number of pixels with the molecule's unique SERS signature intensity I(cm ⁻¹ ). Similar to step (1) of analysis 1, the spectral moment with the largest pixel is used as the background signal and its standard deviation is used to determine the threshold. I(cm ⁻¹ ) threshold=I(cm ⁻¹ ) background+n ^* s. d (cm ^-1 ). Here, n is an integer used as a parameter for adjusting the threshold value. Generally, in this work n is set to 3, 5 or 7.

(2) In order to position a local hot spot, a confocal microscope apparatus is used to raster-scan the entire detection site of the detection plate in the same manner as in (1). Unlike Analysis-1 or Analysis-2, SERS is a label-free detection method and single molecule SERS signals do not show binary behavior. Therefore, the SERS signature I (cm ⁻¹ ) between the individual bright pixels is compared to determine the number of labels at the bright pixels. Due to the large expansion of the detection plate, single or multiple analytes can be distinguished from the background. The quantity of analyte in the hotspot can then be determined.

  (3) After completing (1) and (2), a list of hotspot pixel coordinates, SERS signature intensities and corresponding label numbers can be tabulated. The total number of labels is obtained by SUM for each bright pixel label number.

  Analysis-4 Method Analysis-4 of one embodiment of signal analysis includes (1) taking an image (ie, a picture) of the relevant area of the plate with a smartphone, (2) data locally (same smartphone). Using (analyzing), remotely (sending data to a remote site for analysis), or both, and (3) with or without expert advice on the meaning of the data Including the step of displaying the data on the smartphone in the context. In some embodiments, the analysis includes imaging processing methods, including but not limited to those in Open-CV or Image-J.

14 Labels One or any combination of the embodiments of photolabels described throughout this disclosure applies to all methods and devices described throughout the description of this invention.

  In some embodiments, the label adheres to the detection agent, analyte or entity (connectant). In certain embodiments, the label is a photolabel, an electrical label, an enzyme used to generate an optical or electrical signal, or any combination thereof. In certain embodiments, the detection agent, analyte or entity (connectant) is attached to a connecting molecule (eg, protein, nucleic acid, or other compound) that subsequently adheres to the label.

  In some embodiments, the optical beacon is an object capable of producing a light signal, the generation of the light signal includes reflection of light (ie, photons), scattering, transmission, absorption, spectrum, color, emission, intensity, It includes, but is not limited to, wavelength, position, polarization, luminescence, fluorescence, electroluminescence, photoluminescence (fluorescence), chemiluminescence, electrochemiluminescence, or any combination thereof. In some embodiments, the optical signal is in the form of an optical image (ie, optical signal and sample or device position) or the total number of all photons from a given area or volume. The preferred wavelength of light is in the range of 400 nm to 1100 nm, in the range of 50 nm to 400 nm, in the range of 1 nm to 50 nm, or in the range of 1100 nm to 30,000 nm. Another preferred wavelength is terahertz.

  Beads, Nanoparticles, and Quantum Dots In some embodiments, the photolabel is beads, nanoparticles, quantum dots, or any combination thereof.

  In some embodiments, the diameter of the beads, nanoparticles or quantum dots is 1 nm or less, 2 nm or less, 5 nm or less, 10 nm or less, 20 nm or less, 30 nm or less, 40 nm or less, 50 nm or less, 60 nm or less, 70 nm or less, 80 nm or less. , 100 nm or less, 120 nm or less, 200 nm or less, 300 nm or less, 500 nm or less, 800 nm or less, 1000 nm or less, 1500 nm or less, 2000 nm or less, 3000 nm or less, 5000 nm or less, or a range between any two of these values.

  In some embodiments, beads or quantum dots are used as labels, and they are pre-coated on the plates of CROF, and the internal spacing between the two plates is 1 μm or less, 10 μm or less, 50 μm or less, or these A range between any two of the values.

In some embodiments, separation between beads in solution
-Diffusion time. (The relevant volume of transport medium thickness results in a diffusion time of the photolabels across the thickness of less than 1 ms)
-The dissolution time can be controlled. It can be controlled using photons, heat, or other excitations, and combinations thereof. Dissolution does not begin until excitation energy is applied.

  In some embodiments of the label are nanoparticles having a diameter of 10 nm or greater. Such large diameter nanoparticles have smaller diffusion constants than small molecules (mass <1000 Da) and large molecules (mass = 1,000 to 1,000,000 Daltons (da)) and for a given solution and distance. The diffusion time becomes longer. The reduction of the diffusion time is to reduce the diffusion distance.

  In the method of any of paragraphs Q1-Q2, the one or more open configurations include configurations in which the plates are spaced apart from each other such that the sample is attached directly to one plate so that another plate is not present.

  Q1. The method of paragraph Q1, wherein the one or more open configurations comprises a configuration wherein the plates are far apart from each other such that the sample adheres to one of the pair of plates without interference with the other pair of plates.

  If the photolabels are beads or other nanoparticles with diameters larger than a few nanometers, they have particular advantages over the prior art. This is because the diffusion constant of an object in a liquid is inversely proportional to the diameter of the object with respect to the first approximation (according to the Einstein-Stokes equation).

  For example, bead photolabels having diameters of 20 nm, 200 nm, and 2000 nm, respectively, have diffusion constants, and thus have diffusion times that are 10, 100, and 1000 times greater than 2 nm beads. This results in longer actual saturation incubation times for PoC (point of care) applications, relative to the typical diffusion distances used in current assays.

  However, the present invention solved the problem of long incubation times for photolabels with diameters larger than a few nanometers. The present invention has photolabels stored on the plate surface and then placed on a storage surface adjacent to binding sites with distinct distances (between two) on the submillimeter, micrometer, or nanometer scale. , Filling the separation gap with the transport medium (the stored photolabel dissolves in the transport medium and diffuses to the binding site). The present invention further allows such small distances to be uniformly controlled over the area of the large binding site, and can be easily controlled by using spacer technology.

  Labeling the analyte may include, for example, using a labeling agent such as an analyte-specific binding member that comprises a detectable label. Detectable labels include, but are not limited to, fluorescent labels, colorimetric labels, chemiluminescent labels, enzyme-linked reagents, multicolor reagents, avidin-streptavidin-related detection reagents, and the like. In certain embodiments, the detectable label is a fluorescent label. A fluorescent label is a labeling moiety that can be detected by a fluorescence detector. For example, the binding of a fluorescent label to the analyte of interest may allow the analyte of interest to be detected by a fluorescence detector. Examples of fluorescent labels include, but are not limited to, fluorescent molecules that fluoresce when contacted with a reagent, fluorescent molecules that fluoresce when irradiated with electromagnetic radiation (eg, UV, visible light, X-rays, etc.). ..

  In certain embodiments, suitable fluorescent molecules (fluorophores) for labeling include IRDye 800CW, Alexa 790, Dylight 800, fluorescein, fluorescein isothiocyanate, succinimidyl ester of carboxyfluorescein, succinimidyl ester of fluorescein, fluoroscein. 5-isomer of dichlorotriazine, caged carboxyfluorescein-alanine-carboxamide, Oregon Green 488, Oregon Green 514, Lucifer Yellow, Acridine Orange, Rhodamine, Tetramethylrhodamine, Texas Red, Propidium Iodide, JC-1 (5. 5′,6,6′-tetrachloro-1,1′,3,3′-tetraethylbenzimidazoylcarbocyanine iodide), tetrabromorhodamine 123, rhodamine 6G, TMRM (tetramethylrhodamine methyl ester), TMRE ( Tetramethylrhodamine ethyl ester), tetramethylrosamine, rhodamine B and 4-dimethylaminotetramethylrosamine, green fluorescent protein, blue-shifted green fluorescent protein, cyan-shifted green fluorescent protein, red-shifted green fluorescent protein, yellow Shifted green fluorescent protein, 4-acetamido-4'-isothiocyanate stilbene-2,2' disulfonic acid, acridine, acridine, acridine and its derivatives such as isothiocyanate, 5-(2'-aminoethyl)aminonaphthalene-1-sulfone. Acid (EDANS), 4-amino-N-[3-vinylsulfonyl)phenyl]naphtho-alimido-3,5 disulfonate, N-(4-anilino-1-naphthyl)maleimide, anthranilamide, 4,4- Difluoro-5-(2-thienyl)-4-bora-3a,4a diaza-5-indacene-3-propioni-c acid BODIPY, Cascade Blue, Brilliant Yellow, Coumarin, 7-Amino-4-methylcoumarin (AMC, Coumarin 120), coumarin and derivatives such as 7-amino-4-trifluoromethylcoumarin (coumarin 151), cyanine dye, cyanocin, 4′,6-diaminidino-2-phenylindole (DAPI), 5′,5″. -Dibromopyrogallol-sulfonaphthalein (bromopyrogallol red), 7-diethylamino-3-(4'-isothiocyanatophenyl)-4-methylcoumarin, diethylenetriamine pentaacetate, 4, 4'-diisothiocyanatodihydro-stilbene-2-,2'-disulfonic acid, 4,4'-diisothiocyanatostilbene-2,2'-disulfonic acid, 5-(dimethylamino]naphthalene-1-sulfonyl Chloride (DNS, dansyl chloride), 4-dimethylaminophenylazophenyl-4'-isothiocyanate (DABITC), eosin and derivatives such as eosin, eosin isothiocyanate, erythrosine B, erythrosine, erythrosine and derivatives such as isothiocyanate. , Ethidium, 5-carboxyfluorescein (FAM), 5-(4,6-dichlorotriazin-2-yl)amino-fluorescein (DTAF), 2',7'dimethoxy-4'5'-dichloro-6-carboxy Fluorescein and its derivatives such as fluorescein (JOE), fluorescein, fluorescein isothiocyanate, QFITC, (XRITC), fluorescamine, IR144, IR1446, malachite green isothiocyanate, 4-methylumbellion-ferroneorthocresolphthalein, nitrotyrosine, Pararosaniline, phenol red, B-phycoerythrin, o-phthaldialdehyde, pyrene, pyrene butyric acid, pyrene and derivatives such as succinimidyl 1-pyrene, butyrate quantum dots, 6-carboxy-X-rhodamine (ROX), 6- Such as carboxyrhodamine (R6G), lissamine rhodamine B sulfonyl chloride rhodamine (Rhod), rhodamine B, rhodamine 123, rhodamine X isothiocyanate, sulforhodamine B, sulforhodamine 101, sulfonyl chloride derivative of sulforhodamine 101 (Texas red) Reactive Red 4 (Cibacron™ Brilliant Red 3B-A) rhodamine and derivatives, N,N,N′,N′-tetramethyl-6-carboxyrhodamine (TAMRA), tetramethylrhodamine, tetramethylfodamine isothiocyanate (TRITC), riboflavin, 5-(2'-aminoethyl)aminonaphthalene-1-sulfonic acid (EDANS), 4-(4'-dimethylaminophenylazo)benzoic acid (DABCYL), rosolic acid, CAL Fluor Orange 560. , Terbium chelate derivative, Cy3, Cy5, Cy5.5, Cy7, IRD700, IRD800, rayola blue, phthalocyanine Nin, and naphthalocyanines, coumarins and related dyes, xanthene dyes such as resorufin, bimane, acridine, isoindole, dansyl dyes, aminophthalhydrazides such as luminol and isoluminol derivatives, aminophthalimide, aminonaphthalimide, aminobenzofuran, Including, but not limited to, aminoquinoline, dicyanohydroquinone, fluorescent europium and terbium complexes, combinations thereof, and the like. Suitable fluorescent and chromogenic proteins are GFPs or derivatives thereof derived from Aequorea esculenta, eg “humanized” derivatives such as enhanced GFP, GFP from different species such as Renilla, Renilla reli, Tyrosarcus gelni, “human”. "Modified" recombinant GFP (hrGFP), including, but not limited to, green fluorescent protein (GFP), including but not limited to any of a variety of fluorescent and colored proteins from flowering species, combinations thereof, and the like. ..

  In certain embodiments, the labeling agent is configured to specifically bind the analyte of interest. In certain embodiments, the labeling agent may be present in the CROF device prior to applying the sample to the CROF device. In other embodiments, the labeling agent may be applied to the CROF device after applying the sample to the CROF device. In certain embodiments, after applying the sample to the CROF device, the CROF device is washed to remove any unbound components such as unbound analyte and other non-analyte components in the sample, and After washing, a labeling agent may be applied to the CROF device to label the bound analyte. In some embodiments, the CROF device is washed after binding the labeling agent to the analyte capture agent complex to remove any excess labeling agent not bound to the analyte-capture agent complex from the CROF device. To do.

  In certain embodiments, after binding the analyte to the CROF device, for example, using the labeled binding agent capable of binding the analyte in the CROF device, eg, in a sandwich-type assay, while at the same time binding the analyte as a capture agent. And label the analyte. In some embodiments, nucleic acid analytes may be captured on a CROF device and labeled nucleic acids can simultaneously hybridize to the analyte as a capture agent to which the nucleic acid analyte is bound to the CROF device. ..

  In certain aspects, the CROF device enhances the optical signal, such as fluorescence or luminescence, produced by the detectable label that is directly or indirectly bound to the analyte and then bound to the CROF device. In certain embodiments, the signal is enhanced by the physical process of signal amplification. In some embodiments, the optical signal is enhanced by the nanoplasmonic effect (eg, surface enhanced Raman scattering). For example, Li et al., Optics Express 2011 19: 3925-3936 and WO 2012/024006 describe examples of signal enhancement by the nanoplasmonic effect and are incorporated herein by reference. In certain embodiments, signal enhancement is achieved without the use of biological/chemical amplification of the signal. Biological/chemical amplification of the signal may include enzymatic amplification of the signal (eg, used in enzyme linked immunosorbent assay (ELISA)) and polymerase chain reaction (PCR) amplification of the signal. In other embodiments, signal enhancement may be achieved by physical processes and biological/chemical amplification.

In certain embodiments, the CROF device is 10 ³ fold or more, eg, 10 ⁴ fold or more, 10 ⁵ fold or more, 10 ⁶ fold or more, 10 ⁷ fold, or more, the signal from a detectable label in proximity to the surface of the CROF device. Conventional nucleic acid microarrays configured to enhance 10 ⁸ fold or more and bound to an analyte on a conventional ELISA plate, ie, compared to a detectable label that is not in close proximity to the surface of a CROF device. Above, the signal is enhanced in the range of 10 ³ to 10 ⁹ fold, for example 10 ⁴ to 10 ⁸ fold, or 10 ⁵ to 10 ⁷ fold, compared to the detectable label as suspended in solution. can do. In certain embodiments, the CROF device is 10 ³ fold or more, eg, 10 ⁴ fold or more, 10 ⁵ fold or more, 10 ⁶ fold or more, 10 ⁷ fold, or more, the signal from a detectable label in proximity to the surface of the CROF device. As a result, the signal is increased by 10 ³ compared to an analyte detection array that is configured to enhance 10 ⁸ fold or more, and as described above, which is not configured to enhance the signal using the physical amplification process. ^{to 109} fold, for ^example, can be enhanced by 10 4 to 10 ⁸ times or ¹⁰ 5 -10 ⁷ fold range.

  Sensitivity In certain embodiments, a CROF device is 0.1 nM or less, such as 10 pM or less, or 1 pM or less, or 100 fM or less, such as 1 fM or less, or 0.5 fM or less, or 100 aM or less, or 50 aM or less, or 20 aM. It is configured to have a detection sensitivity of 10 fM or less including the following. In certain embodiments, the CROF device is configured to have a detection sensitivity of 10aM to 0.1nM, for example 20aM to 10pM, including 100aM to 100fM, 50aM to 1pM. In some cases, the CROF device analyzes at concentrations below 1 ng/mL, such as below 10 pg/mL, below 1 pg/mL, below 100 fg/mL, below 10 fg/mL, or below 100 pg/mL, including below 5 fg/mL. It is configured to detect an object. In some cases, the CROF device is configured to detect an analyte at a concentration of 1 fg/mL to 1 ng/mL, such as 5 fg/mL to 100 pg/mL, including 10 fg/mL to 10 pg/mL. In certain embodiments, the CROF device is configured to have a dynamic range of 5 digits or more, such as 6 digits or more, including 7 digits or more.

  Reading Depending on the case, the period from the application of the sample to the CROF device until the reading on the CROF device is 1 second to 30 minutes, for example 10 seconds to 20 minutes including 1 minute to 5 minutes, 30 seconds to 10 minutes. May be in range. In some cases, the period from application of the sample to the signal enhancement detector to generation of output receivable by the device is 1 hour or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less. 1 minute or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, 1 second or less, or even shorter. In some cases, the time period from application of the sample to the signal-enhanced detector to generation of output receivable by the device is 100 ms or more, including 200 ms or more, eg, 500 ms or more, 1 second or more, 10 Seconds or longer, 30 seconds or longer, 1 minute or longer, 5 minutes or longer, or even longer.

  Any suitable method can be used to read the CROF instrument to obtain a measure of the amount of analyte in the sample. In some embodiments, reading into the CROF device comprises obtaining an electromagnetic signal from a detectable label bound to the analyte within the CROF device. In a particular embodiment, the electromagnetic signal is an optical signal. The resulting optical signal can include light intensity, light wavelength, light source location, and the like. In certain embodiments, the optical signal produced by the label has a wavelength in the range of 300 nm to 900 nm. In a particular embodiment, the optical signal is read in the form of a view of the CROF device.

  In certain embodiments, reading into the CROF device comprises providing an electromagnetic radiation source, such as a light source, as an excitation source for the detectable label attached to the biomarker within the CROF device. The light source may be any suitable light source for exciting the detectable label. Exemplary light sources include, but are not limited to, sunlight, ambient light, UV lamps, fluorescent lamps, light emitting diodes (LEDs), photodiodes, incandescent lamps, halogen lamps, and the like.

  Reading into the CROF device can be accomplished by any suitable method for measuring the amount of analyte present in the sample and bound to the CROF device. In certain embodiments, the CROF device is read by a device configured to obtain a light signal from a detectable label bound to an analyte within the CROF device. In some cases, the device is a handheld device such as a mobile phone or smartphone. Any suitable handheld device configured to read a CROF device may be used in the devices, systems and methods of the present invention. A device configured to read a CROF device is described in US Provisional Patent Application No. 62/066,777, filed October 21, 2014, which application is incorporated herein by reference.

  In some embodiments, the device includes an optical recording device configured to acquire an optical signal from the CROF device, eg, an image of the CROF device. In some cases, the optical recording device is a camera, such as a digital camera. The term "digital camera" is any camera that includes as its main components an imaging lens system for forming an optical image, an image sensor for converting the optical image into an electrical signal, and an imaging device with other components. , Examples of such cameras are digital still cameras, digital movie cameras, and webcams (ie those connected directly to the network and those connected to the network via a device such as a personal computer with information processing capabilities). Camera, publicly or privately connected to a device connected to the network in order to allow the exchange of images, including both images. In one example, the reading on the CROF device may include video imaging, which may capture changes over time. For example, a video may be acquired to provide an assessment of the dynamic changes of the sample applied to the CROF device.

  In certain embodiments, the optical recording device has a lower sensitivity than that of the high sensitivity optical recording device used in research/clinical laboratory equipment. In some cases, the optical recording device used in this method may be less than 1/200, 1/500, 1/1000 or less than the sensitivity of the high-sensitivity optical recording device used in the research/clinical laboratory device. It has a sensitivity of 1/10 or less such as 1/100 or less.

  In certain embodiments, the device may have a video display. A video display is a component by which a display page may be displayed in a user-perceptible manner, such as a computer monitor, cathode ray tube, liquid crystal display, light emitting diode display, touch pad or touch screen display, and/or visually perceptible. Other means known in the art for producing the possible outputs may be included. In certain embodiments, the device displays information such as a report generated from images acquired from the detector and/or processed data and a touch to allow the information to be entered by the subject. Equipped with a screen.

15 Multiplexing In any of the embodiments described herein, the system may be designed to perform multiplex assays, and may have multiple storage sites, multiple binding sites, or multiple storage sites and multiple storage sites. By including a binding site, different assays can be performed in different areas on the surface of one plate. For example, in one embodiment, one plate may contain multiple binding sites, each containing a different capture agent, thus allowing detection of multiple analytes in a sample in the same assay. These sites are proximal to each other, but may be spatially separated from each other.

  FIG. 10 schematically illustrates an exemplary embodiment of the invention, ie, multiplex detection in a single CROF device using one binding site on one plate and multiple storage sites on another plate. Figures (a) and (b) are perspective and cross-sectional views, respectively, of an exemplary device. In an exemplary case, the multiplexed CROF device comprises a first plate and a second plate, one surface of said first plate having one binding site, and one surface of said second plate One surface has multiple storage sites, and different storage sites can have the same detection agent with different concentrations, or different detection agents with the same or different concentrations. In some embodiments, the binding site area is greater than the area of each storage site. In some embodiments, the area of the binding site is greater than the total area of all storage sites and/or the binding sites are aligned with the storage sites (ie they are on top of each other, ie The shortest distance between the binding site and a point on the reservoir is the same or about the same).

  FIG. 11 schematically illustrates a further exemplary embodiment of the present invention, namely multiplex detection in a single CROF device using one storage site on one plate and multiple binding sites on another plate. Figures (a) and (b) are perspective and cross-sectional views, respectively, of an exemplary device. In an exemplary case, the multiplexed CROF device includes a first plate and a second plate, one surface of the first plate having a plurality of binding sites and a second plate of the second plate. One surface has one storage site and different binding sites can have the same capture agent with different concentrations, or different capture agents with the same or different concentrations. In some embodiments, the area of the storage site is larger than the area of each binding site. In some embodiments, the area of the storage site is greater than the total area of all binding sites and/or the storage sites are aligned with the binding sites (ie they are on top of each other).

  FIG. 12 schematically illustrates a further exemplary embodiment of the present invention, ie, multiplex detection in a single CROF device using multiple binding sites on one plate and multiple corresponding storage sites on another plate. Show. Figures (a) and (b) are perspective and cross-sectional views, respectively, of an exemplary device. In an exemplary case, the multiplexed CROF device includes a first plate and a second plate, one surface of the first plate having a plurality of binding sites and a second plate of the second plate. One surface has a plurality of corresponding storage sites, each corresponding storage site being located at a position on the second plate that corresponds to the position of the binding site on the first plate, so that the plate is When placed facing each other, each binding site only overlaps one storage site, each storage site only overlaps one storage site, and different storage sites can have the same detection agent with different concentrations? , Or may have different detection agents with the same or different concentrations, different storage sites may have the same capture agent with different concentrations, or have different capture agents with the same or different concentrations be able to.

  In certain embodiments, in the device of any of FIGS. 10, 11 and 12, the first plate further comprises a first predetermined assay site and a second predetermined assay site on its surface, The distance between the edges proximate to the assay site is substantially greater than the thickness of the uniform thickness layer when the plate is in the closed position, and at least a portion of the uniform thickness layer of the sample has a predetermined thickness. Located above the assay site of the sample and the sample has one or more analytes capable of diffusing into the sample. Since the saturation incubation of the assay can be completed during the significant interdiffusion between two adjacent sites, increasing the distance between the edges of adjacent multiple assay sites to be greater than the thickness of the sample It is possible to have multiple binding sites without fluidly isolating different parts of the. By appropriately selecting the ratio of adjacent distance to the thickness of the sample, and by appropriately selecting a measurement time that is longer than the assay saturation incubation time but shorter than the time of significant interdiffusion between two adjacent sites, Multiplexing can be done by CROF without separating different parts of the sample. In some embodiments, the ratio of adjacent distance to sample thickness in the closed configuration is 1.5 or greater, 3 or greater, 5 or greater, 10 or greater, 20 or greater, 30 or greater, 50 or greater, 100 or greater, 200 or greater, 1000 or more, 10000 or more, or a range between any two of these values. This ratio is 3 or more in preferred embodiments, 5 or more in other preferred embodiments, 10 or more in certain preferred embodiments, 30 or more in other preferred embodiments, and another preferred embodiment. It is 100 or more in the form.

  In a particular embodiment, in the device of any of FIGS. 10, 11 and 12, the first plate has at least three analyte assay sites on its surface, and any two adjacent assay sites. The distance between the edges is substantially greater than the thickness of the layer of uniform thickness when the plate is in the closed position, at least a portion of the layer of uniform thickness located above the assay site and Have one or more analytes that can diffuse into the sample.

  In a particular embodiment, in the device of any of FIGS. 10, 11 and 12, the first plate has a surface substantially less than the thickness of the layer of uniform thickness when the plate is in the closed position. Having at least two adjacent analyte assay sites that are not separated by a large distance, at least a portion of the layer of uniform thickness is located above the assay sites, and the sample is one or more capable of diffusing into the sample. With analyte.

  Any of paragraphs U1-6, X-6, P1-8, W1-6, V1-4, UAB1-8, M1-2, S1-2, Q110, and H1, and any combination thereof, or In the device, the first and second plates further include a binding site and a storage site, as described in FIG. 10, 11 or 12 for multiplexed detection.

  In these embodiments, the device can be for parallel multiplexed assays of liquid samples without fluid separation (ie, there is no physical barrier between assay areas). The device includes a first plate and a second plate, i. The plates are movable relative to each other in different configurations, one or two plates being flexible, ii. One or two plates including spacers secured to each plate, and the spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers, iii. Each plate has a sample contacting area on its respective surface for contacting a sample containing a sample containing one or more target analytes capable of diffusing into the sample, iii. The first plate has on its surface one or more binding sites each having a predetermined region containing a capture agent that binds to and immobilizes the corresponding target analyte of the sample, and iv. The second plate has one or more corresponding storage sites on its surface, each storage site having a predetermined area, and upon contact with the sample dissolves in the sample and diffuses into the sample. Containing a concentration of detection agent, each capture agent, target analyte, and corresponding detection agent are capable of forming a capture agent-target analyte-detection agent sandwich at the binding site of the first plate in one configuration. Is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two of the plates, another configuration is an open configuration A closed configuration configured after depositing the sample in the configuration, and wherein i. At least some of the samples are compressed into a layer of uniform thickness, said layer being in contact with and defined by the inner surfaces of the two plates, and having one or more binding sites and one or more Coating the storage site, ii. One or more corresponding storage sites are above the one or more binding sites, and iii. The uniform thickness of the layer is adjusted by spacers and plates, which is less than 250 μm, substantially less than the linear dimension of the predetermined area of each storage site, and iv. There is no fluidic separation between the binding sites and/or storage sites, and the separation between the edges of adjacent storage sites and the separation between the edges of adjacent binding sites is related to the target analyte or detection agent. There is more than a diffusible distance within a period of time and there is no fluidic separation between the binding and/or storage sites.

  In some embodiments, the first plate has multiple (at least 2, at least 4, or at least 16 or more) binding sites on its surface.

  In some embodiments, each binding site binds to a different target analyte.

  In some embodiments, the second plate has a plurality of (at least 2, at least 4, or at least 16 or more) corresponding storage sites on its surface.

  In some embodiments, each corresponding storage site is bound to a different target analyte.

  In some embodiments, the first plate has a plurality of binding sites on its surface and the second plate has a plurality of corresponding storage sites on its surface such that the plate is in a closed configuration. At some time each binding site opposes a corresponding storage site.

  In some embodiments, the first plate has a plurality of binding sites on its surface and the second plate has a storage site on its surface when the plate is in the closed configuration. At least some of the binding sites face a region in the storage site.

  In some embodiments, the first plate has one binding site on its surface and the second plate has multiple storage sites on its surface when the plate is in the closed configuration. At least some of the storage sites face one region in the binding site.

  In some embodiments, the first plate has a plurality of binding sites on its surface, the binding sites comprising different capture agents that bind to and immobilize the same target analyte.

  In some embodiments, the first plate has a plurality of binding sites on its surface, the binding sites comprising the same capture agent.

  In some embodiments, the capture agents differ in density at different binding sites. These embodiments may be used to provide a method of quantifying the amount of analyte in a sample.

  In some embodiments, there is a separation between two adjacent binding sites or two adjacent storage sites and the ratio of separation to sample thickness in the closed configuration is at least 3, eg at least 5 , At least 10, at least 20, or at least 50.

  In some embodiments, the distance between the spacers is in the range of 1 μm to 120 μm.

  In some embodiments, the thickness of the flexible plate is in the range of 20 μm to 250 μm (eg, in the range of 50 μm to 150 μm) and the Young's modulus is in the range of 0.1 GPa to 5 GPa (eg, 0 GPa. 0.5 GPa to 2 GPa).

  In some embodiments, the thickness of the flexible plate times the Young's modulus of the flexible plate is in the range of 60 GPa-μm to 750 GPa-μm.

  In some embodiments, the method comprises: (a) obtaining a sample containing one or more target analytes that can diffuse into the sample, (b) moveable relative to each other in different configurations. Obtaining a first plate and a second plate, i. One or two plates including spacers fixed to the respective plates, the one or two plates being flexible, ii. The spacers have a predetermined substantially uniform height and a predetermined constant distance between the spacers, iii. The first plate has on its surface one or more binding sites each having a predetermined region containing a capture agent that binds and immobilizes the corresponding target analyte of (a), and iv. The second plate includes one or more corresponding storage sites on its surface, each storage site having a predetermined area, which upon contact with the sample dissolves in the sample and diffuses into the sample. A concentration of detection agent, each capture agent, target analyte, and corresponding detection agent capable of forming a capture agent-target analyte-detection agent sandwich at the binding site of the first plate, step (c) plate Attaching the sample to one or two plates when the is configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and the distance between the plates is adjusted by a spacer. Unconfigured step, (d)(c), followed by compressing the sample by placing the two plates in a closed configuration, the closed configuration comprising: i. At least a portion of the sample is compressed into a layer of uniform thickness, said layer contacting and defined by the inner surfaces of the two plates, the one or more binding sites and one or more storages. Touching the site, ii. One or more corresponding storage sites are on the one or more binding sites, and iii. A uniform thickness of the layer is adjusted by spacers and plates, less than 250 μm, substantially less than the linear dimension of the predetermined area of each storage site, step (e) after (d) and plate While in a closed configuration, (1) incubating the sample for a relevant length of time and then stopping the incubation, or (2) for a time greater than or equal to the minimum relevant length of time. And then assessing the binding of each target analyte to the binding site within a time less than or equal to the maximum of the relevant length of time, the relevant length of time being i. Greater than or equal to the time required for the target analyte in (a) to diffuse over the thickness of the layer of uniform thickness in the closed configuration, and ii. Significantly shorter than the time required for the target analyte in (a) to laterally diffuse over the smallest linear dimension of a given region of the storage or binding site, thereby causing a reaction, in which the reaction (1 ) At the end of the incubation or during the evaluation in (2), the majority of the capture agent-target analyte-detection agent sandwich bound to each binding site is from the corresponding relevant volume of sample and , Each target analyte is allowed to bind to the binding site and the detection agent, and the corresponding relevant volume is the portion of the sample at the corresponding storage site in the closed configuration, between the edges of adjacent storage sites. And the separation between adjacent edges of the binding site is greater than the distance over which the target analyte or detection agent can diffuse within the relevant time period, and the fluidic separation between the binding and/or storage sites. It may include steps that are not present.

  Any embodiment of the multiplex assay device described above may be used in this method.

16 Assay/Chemical reaction (E) of small amount of sample or reagent in wide well
In some applications, the wells on the plate are used to test a sample that has a small sample volume for the area of the well that the sample must cover. One aspect of the present invention is a method and apparatus that allows assaying and other chemical reactions of small amounts of samples or reagents in wide wells. The term "well" refers to a hollow compartment, recessed area, or depression at a surface that prevents liquids deposited inside the well from flowing outside the well by the solid bottom and sealed sidewalls of the well. (FIG. 8). The well region is the region surrounded by the sidewalls. The terms "small volume sample" and "broad well" refer to the volume of a sample on the well bottom having a contact area with the well bottom when the sample is dropped on the bottom of the well and there is no device for dispersing the sample. , Smaller than the area of the well (ie, smallness and breadth are a comparison of the natural contact area of the sample and the bottom area of the well). The well acts as a closed spacer (E).

  8 and 9 show specific embodiments of plates and sealing spacers (wells) for sample thickness adjustment. (A) the first plate has one closed spacer (well) and at least one spacer inside the well (FIG. 9), and (b) the first plate has no spacer inside the well. (FIG. 8), two exemplary embodiments are shown. Another embodiment is that in a closed configuration of plates, the closed spacers are on one plate therein and the isolated spacers are on another plate before the first and second plates are in the closed configuration. , The isolated spacer is inside the well.

  In one embodiment, the volume of sample attached to the wells of the plate is a predetermined volume (the product of the inner well area and the height of the well) that is approximately equal to the internal volume of the well (which measures the volume to a particular volume). , So that when the plate is in the closed configuration, the sample fills the well almost completely, with no or almost no sample flowing out of the well.

  In another embodiment, the volume of sample attached to the wells of the plate is not measured while the other portion of the sample flows out of the well, and in the closed configuration of the plate some of the sample fills the well almost completely. ..

  In another embodiment, the wells are a plate. In some embodiments, there are grooves (damping spaces) between the wells for samples that overflow the wells. The damping space prevents the sample overflowing from one well from flowing to the other well.

E1. A method for assaying and/or a chemical reaction in a small number of samples in a wide well, as shown in FIG.
(A) Obtaining a first plate and a second plate that are movable relative to each other in different configurations, said first plate having a predetermined dimension (well bottom, depth and An edge) and a binding site at the bottom of the well.
(B) obtaining a sample, the sample comprising (i) a target entity capable of binding to a binding site and diffusing in the sample, and (ii) a well without contact with a second plate Having a volume and wetting property such that the contact area of the sample deposited only on the bottom of the well is smaller than the area at the bottom of the well,
(C) depositing the sample in the well or on the corresponding area of the second plate or both when the plate is configured in an open configuration, wherein the open configuration comprises two plates Or completely separated and the spacing between the second plate and the bottom of the well is not controlled by the edge of the well (ie, the depth of the well), a step,
(D) after (c) the step of spreading the sample in the plate in a closed configuration, wherein the second plate is coated on the well and the sample on the binding site is the spacer and the second plate in the closed configuration. And the area of contact between the sample and the bottom of the well is greater than when the plate is in the open configuration, including
The corresponding area of the second plate is the area inside the top of the well and the edge of the well in the closed configuration.

  In the method of paragraph E1, the plate further comprises at least isolated spacers within the wells (ie, well spacers).

  In the method of paragraph E1, in some embodiments, the volume of the sample is measured (eg, trying to have a selected volume). The metered volume is about equal to, less than, or greater than the well volume.

  In the method of paragraph E1, in some embodiments the compressive force from outside the plate is configured to hold the plate in a closed configuration.

  In the method of paragraph E1, in some embodiments the capillary force is configured to hold the plate in a closed configuration.

  As shown in FIG. 8d, in the method of paragraph E1, in some embodiments, the bottom of the well, the corresponding region of the second plate, or both are attached with spacers of a predetermined height and in a closed configuration. , The thickness of the sample is adjusted by spacers, edges, or both.

  In some embodiments, the spacer height is equal to, less than, or greater than the well depth. The bottom of the well is flat (ie flat) or curved. In some embodiments, the spacers (1) have a predetermined distance between the spacers, (2) are inside the sample, (3) are fixed to their respective plates, or any combination thereof. Is.

  In some embodiments, the sample volume is approximately equal to the well volume minus the spacer volume. In some embodiments, the second plate is configured to seal the well.

  In some embodiments, the ratio of the well region to the well depth square is 3 or more, 5 or more, 10 or more, 20 or more, 30 or more, 50 or more, 100 or more, 200 or more, 1000 or more, 10000 or more, or these. Is a range between any two values of.

  The ratio of well area to well depth square is 3-20 in the preferred embodiment, 20-100 in another preferred embodiment, 100-1000 in another preferred embodiment, another preferred embodiment. At 1000 to 10,000.

  Device description and requirements are required. The volume is small and unknown.

17 Accurate quantification by correcting for effects caused by non-sample volume (C)
In the CROF process, the sample is always mixed with a non-sample volume due to non-sample objects including, but not limited to, spacers, bubbles, dust or any combination thereof. Bubbles or dust may be introduced using sample attachment or other processes in the CROF process. These sampleless objects occupy the volume that needs to be modified when determining the relevant volume of the sample (the volume of interest) and are within the sample. One aspect of the invention is to compensate for the effects produced by the non-sample volume in the sample of the relevant volume between the two plates, the thickness of the relevant volume being adjusted by the spacer. .

C1. A method for correcting the effects caused by non-sample material in determining the relevant volume of a sample between two plates, comprising:
(A) obtaining a sample in which the relevant volume of the sample is quantified,
(B) Obtaining two plates that are movable relative to each other in different configurations, wherein one or two of the plates have a predetermined spacer distance and height, respectively. Step, including a spacer fixed to the plate of
(C) depositing the sample on one or two of the two plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and A configuration in which the spacing between the plates is not adjusted by spacers, steps,
(D) After (c), the steps of bringing the plates into a closed configuration, wherein the plates face each other and the spacer and an associated volume of sample are located between the plates and the associated volume is The thickness of the sample is adjusted by plates and spacers, and in the open configuration of the plate, the thickness is less than the maximum thickness of the sample, and the associated volume can include the volume of non-sample material,
(E) while the plate is in the closed configuration, (i) measuring the side area of the sample of the relevant volume and (ii) the volume of the non-sample material; and (f) the relevant spacer adjusted by the spacer. Calculating the relevant volume of the sample using the thickness of the volume to correct for the effects of non-sample material,
The relevant volume is at least a portion of the total volume of the sample, the non-sample material is material that is not derived from the sample,
Non-sample volume measurement is by imaging the sample between two plates.

High-precision quantification by double checking of 18 intervals In a CROF, for a given set of conditions, even if the spacer and plate can give a given sample thickness in a closed configuration, during a certain CROF In addition, the actual set of conditions may differ from what is expected, resulting in a given final sample thickness error. To reduce such errors, one aspect of the invention is to double check the final sample thickness in the closed configuration.

C2. A method for determining and checking the thickness of a sample of related volume between two plates, comprising:
(A) obtaining a sample in which a relevant volume of sample is quantified,
(B) Obtaining two plates that are movable relative to each other in different configurations, wherein one or two of the plates have a predetermined spacer distance and height, respectively. Step, including a spacer fixed to the plate of
(C) depositing the sample on one or two of the two plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated and A configuration in which the spacing between the plates is not adjusted by spacers, steps,
(D) After (c), the steps of bringing the plates into a closed configuration, wherein the plates are facing each other and the spacer and an associated volume of sample are located between the plates and the associated volume is The thickness of the sample is adjusted by plates and spacers, and in the open configuration of the plate, the thickness is less than the maximum thickness of the sample, and the associated volume can include the volume of non-sample material,
(E) while the plate is in the closed configuration, (i) measuring the side area of the sample of the relevant volume, and (ii) the volume of the non-sample material, and (f) correcting the effects of the non-sample material. By calculating a sample of relevant volume by
The method wherein the volume of interest is at least a portion of the total volume of the sample and the non-sample material is material that is not derived from the sample.

19 Washing (WS)
In the present invention, one or any combination of the plate press and holding embodiments described herein is used in all methods and devices described throughout the description of the invention.

A method for a wash step in an assay comprising (a) performing steps in one or any combination of the above methods, and (b) rinsing sample or transfer medium between plates.

  In the method using CROF, the plate is washed by holding it in a closed configuration.

  In the method using CROF, wash by separating the plates in a closed configuration.

Sponge 20 Assay using multiple steps (MA)
In the present invention, the embodiments described by this disclosure (ie, all sections) include (a) combining one embodiment with another, (b) using the same embodiment more than once. That is, (c) (a) and (b) can be used in any combination.

MA1.
(A) obtaining a sample containing the analyte,
A method for assaying an analyte in a sample comprising: (b) performing a method that uses CROF; and (c) releasing the plate and performing a method that uses CROF.

  The method of paragraph MA1 further comprises, in some embodiments, after step (c) of MA1 the step of repeating all of the same steps of the method of MA1 at least once.

MA2. ,
(A) obtaining a sample containing the analyte,
(B) performing the method using CROF,
A method for assaying an analyte in a sample comprising the steps of (c) releasing the plate and carrying out a method using CROF (washing), and (d) carrying out a method using CROF.

  In the method of paragraph MA2 in some embodiments, it further comprises, after step (d) of MA2, repeating the same steps of all steps in the method of MA2 at least once.

  The method of paragraph MA2 further comprises, in some embodiments, after step (c) of MA2, repeating the same steps of all steps in the method of MA1 at least once.

MA3.
A first CROF device using a CROF;
Assaying an analyte in a sample comprising one of the plates of the first CROF device and a third plate that forms a second CROF device when the plates of the first CROF device are separated Kit for.

MA4.
A first CROF device using a CROF;
At least one binding site or storage site on the sample contact area of the plate of the CROF device;
A third plate which, when the plates of the first CROF device are apart, combines with one of the plates of the first CROF device to form a second CROF device,
The binding site binds the target analyte to the surface of the plate and the storage site has a reagent capable of dissolving in the sample and diffusing into the sample upon contact with the sample, for assaying the analyte in the sample Kit.

  Imaging involves the use of smartphones. The methods in this section further include the step of illuminating with a light source. The light source may be a laser, LED, lamp, or camera flashlight.

Kit for performing an assay for detecting a target entity in a sample (MQXA)
a. A first plate capable of immobilizing a target entity, wherein one surface of the first plate has one or more binding sites with binding partners that bind to the target entity;
b. A cover plate,
c. A sample in an internal space between the cover plate and the first plate, the sample including the target entity movable in the sample, wherein the shape of the sample is deformable, The two plates are movable with respect to each other, the shape of the sample substantially conforms to the inner surface, at least part of the sample comes into contact with the binding site and the inner spacing is smaller than a certain distance during the incubation, A sample in contact with the binding site,
d. An imaging device capable of imaging the surface of the first plate and/or the surface of the cover plate;
e. A kit for assaying a target entity in a sample, which comprises a measuring device capable of measuring the distance between internal spaces.

  The methods in this section include the use of smartphones. The methods in this section include the use of lighting devices. Illumination devices include lasers, LEDs, lamps, or camera flashlights.

21 Plate Press and Hold (H)
Compressive Force In the CROF process, force is applied to compress the two plates to bring the plates from the open configuration to the closed configuration. The compressive force reduces the spacing between the inner surfaces of the plates, thereby reducing the sample thickness between the plates. In the present invention, compressive forces include, but are not limited to, mechanical forces, capillary forces (due to surface tension), electrostatic forces, electromagnetic forces (including light), and any combination thereof.

  In some embodiments where the plates are moved from an open configuration to a closed configuration, an external force is applied to push the first plate and the second plate toward each other.

  In some embodiments in which the plates are in an open to a closed configuration, an external force is applied to the outside of the first plate and the second plate to urge the plates toward each other, the pressure being higher than the pressure in the plates. A device is used to increase the pressure outside the plate above the pressure inside the plate. This device is limited to sealing devices only.

  In some embodiments, the pressure is provided at least in part by a capillary force due to the interaction between the liquid and the corresponding surface tension between the first plate and the second plate and the plate. In some embodiments, the liquid is the sample itself or the sample mixed with the liquid. In certain embodiments, capillary forces are used with other forces. Often, the sample is often in a liquid and surface tension is suitable for inserting capillary forces. In some embodiments, the deformation of the sample by the plate can be stopped automatically when the capillary force is equal to the force required to deform the sample.

  In certain embodiments, the pressure between the first plate and the second plate (internal pressure) is isolated from the outside of the plate (external pressure), and then the internal pressure is lower than the external pressure, thereby compressing Forces (and thereby sample deformation) are generated. Isolation can be done using a vacuum seal or other device.

  In some embodiments, it is a combination of the above methods.

  Stepwise Press In certain embodiments, the compressive force that causes the plate to be in a closed configuration is applied in a process called a "stepwise press," which first applies the press (ie, applies pressure) to one location on the plate, Thereafter, it includes stepwise addition to other positions of the sample. In some embodiments of the graduated press, after deforming the sample at a location to a desired thickness, the compressive force at that location (excluding the capillary force of the sample itself) is (i) Maintained in the overall process and (ii) removed while pressed into another location, or (iii) used in some parts of the plate in (i) and used in some parts of the sample in (ii) It

  In one embodiment of the stepwise press, rollers are used to move a first plate and a second plate (where the sample is between the plates and the plates are slightly flexible) to another roller or Press against a flat surface.

  In another embodiment, the human finger is a tool for pressing the plate (and thus the sample). Pressing is the contact of part of a person's hand with other parts of the human body (including other parts of the person's hand) or with the person's hand contacting objects (eg, table surfaces). In one embodiment, the press starts at one location on the sample and moves gradually to another location on the sample.

  In one embodiment of the staged press, the pressed air jet is initially at the position (eg, center) of the plate pair (between the first plate and the second plate, one plate of which is slightly Being flexible), the compressive force is gradually expanded to another part of the plate pair.

  In another embodiment, one or two of the first plate and the second plate is flexible and contacts a location on the sample, after which capillary forces at that location deform the sample. Pull the plate pairs together (towards each other).

  The advantage of stepwise pressing is that it allows the sample to be deformed with a small force (since the force is the same, the smaller the pressing area, the greater the pressure), the movement (deformation) of the sample, and/or Including helping to reduce air bubbles in the sample. The greater the pressure, the greater the deformation of the sample. Gradual pressing can improve the thickness uniformity of the deformed sample.

  Pressing device The device for determining the compressive force for sample deformation in CROF has various implementations. In some embodiments, pressing is done with a human hand, for example with a human finger. In certain embodiments, the pressing device includes a human hand, a mechanical clip, a mechanical press, a mechanical clamp, a mechanical slider, a mechanical device, an electromagnetic device, a surface rolling roller, two opposing rollers, a fluid press, Including, but not limited to, hydraulic devices, or any combination thereof. In certain embodiments, pressurized liquid (including pressurized air) is used to directly or indirectly press the first plate and/or the second plate. “Directly” refers to adding the pressurized liquid directly to the first plate and/or the second plate, and “indirect” refers to adding by the third object. Particular embodiments of the press use a combination of the above embodiments of pressing apparatus and methods.

  Also, in certain embodiments of sample deformation, press and sample deformation are monitored. Monitoring can be used to control press and sample deformation. Deformation monitoring includes, but is not limited to, mechanical methods, electrical, optical, chemical, magnetic, and any combination thereof. Mechanical methods include, but are not limited to, mechanical gauges, spacers (mechanical stoppers, described in detail below) and acoustic waves.

  In CROF, the spacing controller is a mechanical press, a mechanical moving stage, a human finger, a liquid that provides a capillary force that pulls the plates toward each other, a liquid that exerts pressure on the plates (including air), or a combination thereof. including.

  In certain embodiments, mechanical (translational and/or rotational) mechanical stages are used to control sample deformation and sample thickness, and work with a monitoring system.

  In some embodiments, at least some of the compressive force is provided by a press (a device that transforms the plates into the closed configuration) configured to attach the plates together to form the closed configuration.

  In some embodiments, humans press the plate. The person may be the person being tested or the person performing the test, or the person taking the sample.

  In some embodiments, pressing the plates is the use of capillary forces to hold the two plates together. Capillary forces are created by rendering at least a portion of the inner surface of one or two plates hydrophilic. Even with removal of some or all of the forces (excluding capillary forces) to press the plates into the closed configuration, the two plates are initially the same plates as when the plates were in the closed configuration, with proper capillary forces. The thickness of the relevant volume of sample, which is the same as the spacing, can be retained.

  In some embodiments, the apparatus for applying a compressive force to the outer surface of the plate to reduce the spacing of the inner surface of the plate includes a contact surface adapted to the outer surface of the plate, the contact surface of the apparatus being the outer surface of the plate. "Compatible with the outer surface of the plate", which is the surface of the device that contacts the surface, refers to the surface of the device deforming during compression so that its shape matches the shape of the outer surface of the plate. In one exemplary embodiment, the compression device refers to the human finger. In another exemplary embodiment, the compression device has a contact surface made of soft plastic or rubber.

  Self-Holding (Maintaining Final Sample Thickness After Removing Compressive Force) In some embodiments of pressing the CROF, some compressive force is removed after the sample is deformed in the closed configuration and the sample Retains the same final sample thickness as when compressive forces are present. This situation is called "self-holding". One reason for self-holding is that after removing the compressive force inserted from the outside of the plate pair, there are still other forces between the inner surfaces of the plates, such as capillary forces, to hold the plate pair together. It is to be. Capillary force is due to the wetting properties of the sample on the plate.

  To have self-holding, it is necessary to control the wetting properties of the surface of the plate, the total contact area of the sample with the plate, the final sample thickness in the closed configuration, or a combination thereof.

  In some embodiments for achieving self-retention, one or two of the inner surfaces of the plate is hydrophilic. That is, either one of the plates has a hydrophilic inner surface, or two of the plates have a hydrophilic inner surface.

  The capillary force depends on the radius of curvature of the liquid surface, and the smaller the curvature, the higher the capillary force. By using a small spacing between the two plates (ie plate pairs), and thus sample thickness, a small curvature can be achieved. In some embodiments, the final sample thickness to achieve self-holding is 10 nm or less, 100 nm or less, 100 nm or less, 500 nm or less, 1 μm (micrometer) or less, 2 μm or less, 3 μm or less, 5 μm or less, 10 μm or less. , 20 μm or less, 50 μm or less, 70 μm or less, 100 μm or less, 150 μm or less, 300 μm or less, 500 μm or less, 700 μm or less, 1000 μm or less, 1200 μm or less, or a range between any two of these values.

In some embodiments, the area of the sample in contact with the plate for self-holding the maximum 10 [mu] m ^2, a maximum 100 [mu] m ^2, a maximum 200 [mu] m ^2, a maximum 500 [mu] m ^2, the maximum 1000 .mu.m ^2, up to 2000 .mu.m ^2, up to 5000 .mu.m ^2, maximum 8000μm ² , maximum 0.01 mm ² , maximum 0.05 mm ² , maximum 0.1 mm ² , maximum 0.5 mm ² , maximum 1 mm ² , maximum 5 mm ² , maximum 10 mm ² , maximum 50 mm ² , maximum 100 mm ² , maximum 500 mm ² , A maximum of 1000 mm ² , a maximum of 2,000 mm ² , a maximum of 5,000 mm ² , a maximum of 10,000 mm ² , a maximum of 100,000 mm ² , or a range between any two of these values.

  In some embodiments, the wetting characteristics of one or two of the inner surfaces of the plate are modified to provide better self-holding.

  HS. 1 In some embodiments, in a CROF process, an apparatus is used to insert a compressive force to bring the plate into a closed configuration, upon which the compressive force by the apparatus is removed and the sample thickness and plate The surface spacing is approximately the same before the compressive force by the device is removed. In some embodiments, the method of the preceding paragraph comprises reading a signal from the plate or between the plates, where the signal is the analyte, entity, label, sample volume, concentration of the substance (ie, chemical), or those , But is not limited to.

  Paragraph SH. In one method, the device is a human hand, a mechanical clip, a mechanical press, a mechanical clamp, a mechanical slider, a mechanical device, an electromagnetic device, a surface rolling roller, two opposing rollers, a fluid press, a hydraulic device. Or any combination thereof.

  Paragraph SH. In one method, in some embodiments, "the thickness of the sample and the inner surface spacing of the plate are about the same as before removal of the compressive force by the device" means that the sample thickness and plate before and after compressive force removal Relative difference of inner surface spacing is 0.001% or less, 0.01% or less, 0.1% or less, 0.5% or less, 1% or less, 2% or less, 5% or less, 8% or less, 10% or less , 15% or less, 20% or less, 30% or less, 40% or less, 50% or less, 60% or less, 70% or less, 80% or less, 90% or less, 99.9% or less, or any of these. Indicates that the range is between the numbers.

  Paragraph SH. In one method, in some embodiments, the sample thickness after removal of the compressive force by the device and the inter-plate surface spacing are predetermined, which is determined before applying the compressive force under a predetermined compressive condition. , Indicates that the thickness and spacing after removal of the compressive force are known.

H1. A method for reducing the thickness of a sample of a relevant volume and retaining the reduced thickness,
(A) obtaining a sample in which the thickness of the relevant volume is reduced
(B) Obtaining two plates that are movable relative to each other in different configurations, wherein one or two of the plates have a predetermined spacer distance and height, respectively. Step, including a spacer fixed to the plate of
(C) depositing the sample on one or two of said plates when the plates are configured in an open configuration, wherein the two configurations are such that the two plates are partially or completely separated and The spacing between is not adjusted by the spacer, is a step,
(D) after (c) the step of spreading the sample using a pressing device in a plate closed configuration, wherein the plates face each other and the spacers and associated volume of sample are applied to the plate. And the thickness of the relevant volume of the sample is adjusted by the plate and the spacer, in an open configuration of the plate, the thickness is less than the maximum thickness of the sample and at least one spacer is located in the sample. After steps, and (e)(d), after releasing the device and releasing the pressing device, the spacing between the plates is kept to be the same or about the same as when the device is applied, Including steps,
The method wherein the relevant volume is at least a portion of the total volume of the sample.

  In the method of paragraph H1, the percentages that are about the same as the spacing between the plates are: max 1%, max 2%, max 5%, max 10%, max 20%, max 50%, max 60%, max 70%, 80% maximum, 90% maximum, or a range between any two of these values.

  For example, in CROF, a human hand compresses the two plates to a closed position and then removes the hand and hand compression forces, but the final sample thickness remains the same when hand compression force is still present. is there.

22 Other Combinations In the present invention, each embodiment of the present disclosure (ie, all sections) is (a) used alone, (b) combined with other embodiments, (c) used multiple times. And any combination of (d)(a) to (c).

  The methods and devices disclosed in the present invention can be used alone or in any combination thereof. The term "QMAX" method or device refers to the method or device of the embodiments described herein.

  In some embodiments, specifically, Q is used for the inventions disclosed in Sections 1 and 2, A is used for the inventions disclosed in Sections 3 and 5, and Section 4 is used. And X for the invention disclosed in Section 5 and M for the invention disclosed in Section 6. Thus, the inventive methods and apparatus disclosed in Sections 1, 2, 3 and 5 are Q, X, A, M, QX, QA, QM, XA, XM, AM, QXA, QAM, XAM, and QXAM. Can be used in form.

a. The first plate surface has at least one well of known depth and volume, and the bottom surface of the well has one or more binding sites capable of immobilizing the target entity in the sample. Having one plate,
b. The sample comprises the target entity, the target entity is moveable within the sample, the shape of the sample is deformable, and the sample covers only a portion of the well (thus providing a thickness greater than the well depth). A), depositing a sample having a volume approximately the same as that of the well in the well,
c. Having a cover plate,
d. The sample is between the inner surfaces of the first plate and the second plate by facing the first plate and the cover plate to each other,
e. Reducing the thickness of the sample by reducing the distance between the inner surfaces of the first plate and the second plate, and f. Some embodiments of the application of Q, X, A, and M to a surface-immobilized assay, including incubating the sample at a reduced sample thickness for a period of time.

  One variation of these methods is to apply one or more of the above steps to a 96 well plate or other plate.

  The methods and devices disclosed in Sections 1, 2, 3, 5 of the present invention can be used alone or in any combination thereof. Specifically, Q is used for the inventions disclosed in Sections 1 and 2, A is used for the inventions disclosed in Sections 3 and 5, and A is used for the inventions disclosed in Sections 4 and 5. We use X for inventions and M for inventions disclosed in Section 6. Thus, the inventive methods and apparatus disclosed in Sections 1, 2, 3 and 5 are Q, X, A, M, QX, QA, QM, XA, XM, AM, QXA, QAM, XAM, and QXAM. Can be used in form.

a. The first plate surface has at least one well of known depth and volume, and the bottom surface of the well has one or more binding sites capable of immobilizing the target entity in the sample. Having one plate,
b. The sample comprises the target entity, the target entity is moveable within the sample, the shape of the sample is deformable, and the sample covers only a portion of the well (thus providing a thickness greater than the well depth). Have),
c. Depositing a sample with approximately the same volume as the well having a cover plate into the well,
d. The sample is between the inner surfaces of the first plate and the second plate by facing the first plate and the cover plate to each other,
e. Reducing the thickness of the sample by reducing the distance between the inner surfaces of the first plate and the second plate, and f. Some embodiments of the application of Q, X, A, and M to a surface-immobilized assay, including incubating the sample at a reduced sample thickness for a period of time.

  One variation of these methods is to apply one or more of the above steps to a 96 well plate or other plate.

  Some embodiments of the methods, devices, and systems described above include sample volume (Q), reagent addition (A) and/or assay acceleration (X) (corresponding acronyms QA, QX, AX, and QAX). ). Described below are some experimental demonstrations of Q, A, X, QA, QX, AX, and QAX methods and apparatus.

23 Reagent Unless otherwise stated, the term “reagent” refers to one or more of biological agents, biochemical agents and/or chemical agents. For example, the reagent may be a capture agent, a detection agent, a chemical compound, a photolabel, a radioactive label, an enzyme, an antibody, a protein, a nucleic acid, a DNA, an RNA, a lipid, a carbohydrate, a salt, a metal, a surfactant, a solvent or any of them. Combinations can be included.

  In some embodiments, the reagents on the plate may be present in the form of liquids, solids, molecular vapors, or a combination thereof. Reagent deposition includes, but is not limited to, deposition, placement, printing, stamping, liquid distribution, evaporation (thermal evaporation, vapor evaporation, human breathing), chemical vapor deposition, and/or sputtering. Different reagents may be in different locations. The reagent may be printed and/or attached with small dots of reagent.

  In some embodiments, the reagents are first applied to the plate in liquid or vapor form and then dried prior to the CROF process to dry reagents on the plate.

  Controlling Reagent Release Time The Method A further comprises controlling the reagent release time (ie, the time taken to measure how fast the reagent dissolves in the sample). Some embodiments of controlling the reagent release time of a reagent include mixing or applying to the top of the reagent one or more "release controlling material" into the sample that affects the reagent release. In some embodiments, the controlled release material may be another reagent. For example, it has two kinds of reagents A and B, the reagent A is applied on the upper surface of the reagent B, and under certain conditions, the reagent A is dissolved in the sample before the reagent B.

  Also, the surface characteristics of the first plate and the second plate can be used to control reagent release. One example is to control the wetting properties of the surface. In many reagents, the hydrophobic surface binds well to the reagent so that the reagent is or is not released into the sample (depending on the thickness of the reagent layer) and the hydrophilic surface is insufficient for the reagent. Due to binding, the reagent is rapidly released into the sample.

The reagents used in the present invention may be any suitable reagents required for the assay, such as labeled or unlabeled antibodies, labeled or unlabeled nucleic acids, enzymes with or without affinity moieties, etc. Good. In some embodiments, as described above, the stored reagent may be an assay component designed to test a blood or other liquid sample for the presence of the analyte. For example, chloride ion can be measured by any of the following methods, and these assay components can be present at the storage site. Colorimetric method: Chloride ion displaces thiocyanate from mercury thiocyanate. Free thiocyanate reacts with ferric ion to form a photometrically measured colored complex-ferric thiocyanate. Coulometric method: A constant direct current flows between silver electrodes to generate silver ions that react with chloride ions to form silver chloride. After all chloride ions have combined with silver ions, free silver ions accumulate and the current between the electrodes increases, indicating the end of the reaction. Mercury method: Chloride ions are titrated with a standard solution of mercury ions to form HgCl2 soluble complexes. The end point of the reaction is detected colorimetrically when excess mercury ions bind to the indicator dye diphenylcarbazone to form a blue color. Similarly, calmagito, which turns reddish purple when reacted with magnesium, can be used to colorimetrically measure magnesium, and by a formazan dye test, it reacts with magnesium or binds to magnesium to form a blue complex. It emits at 600 nm when using methylthymol blue which forms Similarly, calcium can be detected by a colorimetric method using O-cresolphthalein which exhibits a purple color when the O-cresolphthalein complex reacts with calcium. Similarly, in the reaction catalyzed by phosphoenolpyruvate carboxylase (PEPC), bicarbonate (HCO 3 ⁻ ) and phosphoenolpyruvate (PEP) are converted to oxaloacetate and phosphate, so that the bicarbonate ion is dichromated. It can be tested by sex. Malate dehydrogenase (MD) catalyzes the reduction of oxaloacetate to malate, with the attendant oxidation of reduced nicotinamide adenine dinucleotide (NADH). This oxidation of NADH results in a decrease in the absorbance of the reaction mixture measured dichroically at 380/410 nm in proportion to the bicarbonate ion of the sample. Blood urea nitrogen is a colorimetric test in which diacetyl or ferrons produce a yellow chromogen containing urea and can be quantified photometrically or by multiple uses of urease, an enzyme that converts urea to ammonia and carbonic acid. Can be detected by, for example, i) decreasing the absorbance at 340 nm when ammonia reacts with α-ketoglutarate, and ii) measuring the rate of increase in conductivity of the solution in which urea is hydrolyzed. It can be assayed. Similarly, creatinine can be measured colorimetrically by treating a sample with an alkaline picrate solution to give a red complex. Further, creatine can be measured using a non-Jaffe reaction that measures the ammonia produced when creatinine is hydrolyzed by creatinine iminohydrolase. Glucose can be measured in an assay in which blood is exposed to an amount of glucose oxidase for a period of time to estimate its concentration. After a certain time, it is possible to remove excess blood and develop color, which allows the glucose concentration to be estimated. For example, glucose oxidase and glucose react to form neo-oxygen, which converts potassium iodide to iodine (in the filter paper) to produce a brown color. Glycosylated hemoglobin concentration is an indirect reading of glucose levels in the blood. When performing a chromatographic analysis on hemolysis of red blood cells, three or more small peaks, designated Hemoglobin A1a, A1b, and A1c, elute before the major Hemoglobin A peak. These "rapid" hemoglobins are formed by the irreversible binding of glucose and hemoglobin in a two-step reaction. Hexokinase is measured in an assay in which glucose is phosphorylated by hexokinase (HK) in the presence of adenosine triphosphate (ATP) and magnesium ions to produce glucose-6-phosphate and adenosine diphosphate (ADP). be able to. Glucose-6-phosphate dehydrogenase (G6P-DH) specifically oxidizes glucose-6-phosphate to gluconate-6-phosphate and simultaneously reduces NAD+ to NADH. The increase in absorbance at 340 nm is proportional to the glucose concentration in the sample. HDL, LDL, triglycerides can be measured using the Abell-Kendall method including color development with Liebermann-Burchard reagent (mixed reagent of acetic anhydride, glacial acetic acid and concentrated sulfuric acid) at 620 nm after hydrolysis and extraction of cholesterol. it can. Fluorescence analysis can be used to determine triglyceride baseline values. After precipitation of apoprotein B-containing lipoproteins in whole plasma (LDL and VLDL) with heparin-manganese chloride, plasma high density lipoprotein cholesterol (HDL-C) is measured in the same manner as plasma total cholesterol. These compounds are also capable of colorimetric detection in enzyme-driven reaction-based assays that quantitate both cholesterol esters and free cholesterol. Cholesterol esters are hydrolyzed to cholesterol via cholesterol esterase and then oxidized by cholesterol oxidase to cholesta-4-en-3-one and hydrogen peroxide. After that, hydrogen peroxide is detected with a highly specific colorimetric probe. Horseradish peroxidase catalyzes the reaction of hydrogen peroxide with the bound probe in a 1:1 ratio. The sample can be compared to known concentrations of cholesterol standards.

  Drying Reagents In some embodiments, after the reagent deposition step (c) and prior to the sample deposition step (d), the A-method dries some or all of the reagent deposited in step (c). The method further includes steps.

  Location of Reagents Reagents can be applied and/or placed on one or two plates. The reagents may be present at storage sites (locations) on the plate, each storage site containing one or more reagents. Different storage sites may contain different reagents, the same reagents or one or more common reagents.

  Control the concentration of added reagent. In some embodiments, the method can further include controlling the concentration of the added reagent by controlling the thickness of the sample on the storage site (ie, the surface having the reagent).

24 Uses, Samples, and More Biochemical/Chemical Biomarkers The uses of the present invention include (a) infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, and organic matter. Detection, purification, and quantification of compounds or biomolecules that correlate with a specific disease stage such as sexual disease, for example, lung disease, renal disease, (b) environment such as water or soil, or biological sample such as tissue or body fluid , Purification and quantification of microorganisms such as viruses, fungi and bacteria, (c) detection and quantification of hazardous wastes that pose a risk to food safety or national security, chemical substances such as B. anthracis or biological samples , (D) quantification of important parameters in medical or physiological monitors such as glucose, blood oxygen level, complete blood count, (e) detection of specific DNA or RNA from biological samples such as cells, viruses, body fluids and Including quantification, (f) sequencing and comparison of gene sequences in DNA in chromosomes and mitochondria for genomic analysis, or (g) detection of reaction products during, for example, synthesis or purification of pharmaceuticals, Not limited to.

  Detection can be performed in various sample matrices such as cells, tissues, body fluids, and feces. The target body fluids are amniotic fluid, aqueous humor, vitreous humor, blood (eg, whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax, chyle, chyme, Endolymph, perilymph, feces, stomach acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, purulent fluid, mucous secretions, saliva, sebum (skin oil), semen , Sputum, sweat, synovial fluid, tears, vomiting, urine and exhaled breath condensate. In some embodiments, the sample comprises human body fluid. In some embodiments, the sample is cells, tissues, body fluids, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, earwax, chyle, chyle. Juice, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal discharge, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, mucous secretions, saliva, sebum, semen, sputum, sweat, lubrication Includes at least one of fluid, tears, vomiting, urine and exhaled breath condensate.

  In some embodiments, the sample is at least one of a biological sample, environmental sample, biochemical sample.

Uses, Samples, and More Biochemical/Chemical Biomarkers The devices, systems and methods of the present invention provide a variety of different methods where it is desired to determine and/or quantify the presence or absence of one or more analytes in a sample. It is used in different areas. For example, the method of the present invention is used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds and the like. The various areas include, but are not limited to, humans, veterinary medicine, agriculture, food, environment, drug testing, and the like.

  In certain embodiments, the methods of the invention are used to detect nucleic acids, proteins, or other biomolecules in a sample. The method can include the detection of a set of biomarkers in the sample, eg, two or more distinct protein or nucleic acid biomarkers. For example, the method may be used for rapid clinical detection of two or more disease biomarkers in a biological sample, such as diagnosing a subject's disease state, or on-going management or treatment of the subject's disease state. . As noted above, communication with a physician or other healthcare provider will ensure that the physician or other healthcare provider is aware and aware of potential concerns, and thus may take appropriate action. Sex can be higher.

  Applications of the devices, systems, and methods of the present invention using a CROF device include (a) infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, and organic disorders such as lung. Detection, purification and quantification of compounds or biomolecules that correlate with specific disease stages such as diseases and renal diseases, (b) environment, such as water and soil, or viruses, fungi and bacteria from biological samples such as tissues and body fluids Detection, purification and quantification of microorganisms such as, (c) detection and quantification of hazardous wastes, chemical substances or biological samples such as B. anthracis, which pose a risk to food safety or national security, (d) glucose, Quantification of important parameters in medical or physiological monitors such as blood oxygen level, complete blood count, (e) detection and quantification of specific DNA or RNA from biological samples such as cells, viruses, body fluids, (f) This includes, but is not limited to, sequencing and comparison of gene sequences in DNA in chromosomes and mitochondria for genomic analysis, or (g) detection of reaction products during, for example, synthesis or purification of pharmaceuticals. Some specific applications of the device, system and method of the present invention are described in more detail below.

  The application of the present invention applies to (a) specific disease stages such as infectious diseases and parasitic diseases, injuries, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, and organic diseases such as lung diseases and renal diseases. Detection, purification and quantification of correlative compounds or biomolecules, (b) Detection, purification and quantification of microorganisms such as viruses, fungi and bacteria from environment such as water, soil, or biological samples such as tissues and body fluids , (C) detection and quantification of hazardous wastes, chemical substances such as B. anthracis or biological samples that pose a risk to food safety or national security, (d) medicines such as glucose, blood oxygen level, and complete blood count, or Quantification of important parameters in physiological monitors, (e) detection and quantification of specific DNA or RNA from biological samples such as cells, viruses and body fluids, (f) DNA in chromosomes and mitochondria for genomic analysis Sequencing and comparison of gene sequences therein, or (g) including, but not limited to, detection of reaction products during synthesis or purification of pharmaceuticals, for example.

  Detection can be performed in various sample matrices such as cells, tissues, body fluids, and feces. The target body fluids are amniotic fluid, aqueous humor, vitreous humor, blood (eg, whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax, chyle, chyme, Endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, purulent fluid, mucous membrane secretion, saliva, sebum (skin oil), Includes but is not limited to semen, sputum, sweat, synovial fluid, tears, vomiting, urine and exhaled breath condensate. In some embodiments, the sample comprises human body fluid. In some embodiments, the sample is cells, tissues, body fluids, feces, amniotic fluid, aqueous humor, vitreous humor, blood, whole blood, fractionated blood, plasma, serum, breast milk, cerebrospinal fluid, earwax, chyle, chyle. Juice, endolymph, perilymph, feces, gastric acid, gastric juice, lymph, mucus, nasal discharge, mucus, pericardial fluid, peritoneal fluid, pleural fluid, pus, mucous secretions, saliva, sebum, semen, sputum, sweat, lubrication Includes at least one of fluid, tears, vomiting, urine and exhaled breath condensate.

  In some embodiments, the sample is at least one of a biological sample, environmental sample, biochemical sample.

  Implementations of the devices, systems, and methods of the present invention include a) a capture agent bound to a target analyte under conditions suitable for obtaining a sample and b) binding of the analyte in the sample to the capture agent. Applying the sample to the CROF device, c) cleaning the CROF device, and d) reading the CROF device to obtain a measurement of the amount of analyte in the sample. In some embodiments, the analyte may be a biomarker, environmental marker or food marker. In some examples, the sample is a liquid sample, a diagnostic sample (eg saliva, serum, blood, sputum, urine, sweat, tears, semen or mucus), river, ocean, lake, rain, snow, sewage, wastewater treatment wastewater. , An environmental sample obtained from agricultural wastewater, industrial wastewater, tap water, drinking water, etc., or a food sample obtained from tap water, drinking water, processed cooked food, cooked food, or unprocessed food, etc. You may.

  In any embodiment, the CROF device may be located in a microfluidic device and the application of step b) may include application of the sample to the microfluidic device comprising the CROF device.

  In any embodiment, the reading step d) can include detection of fluorescent or luminescent signals from the CROF device.

  In any embodiment, the reading step d) may include reading the CROF device using a handheld device configured to read the CROF device. The handheld device may be a mobile phone such as a smartphone.

  In any of the embodiments, the CROF device can include a labeling agent capable of binding to the analyte-capture agent complex on the CROF device.

  In any of the embodiments, the apparatus, system, and method of the invention comprise applying a labeling agent bound to the analyte-capture agent complex on the CROF device to the CROF device during steps c) and d). , And cleaning the CROF device.

  In any embodiment, the reading step d) may include reading the identifier of the CROF device. The identifier may be an optical barcode, a radio frequency ID tag, or a combination thereof.

  In any embodiment, the devices, systems and methods of the present invention apply a control sample containing a known detectable amount of an analyte to a control CROF device that includes a capture agent that binds to the analyte. Reading the CROF instrument to obtain a control measurement of a known detectable amount of analyte in the sample.

  In any embodiment, the sample may be a diagnostic sample obtained from the subject, the analyte may be a biomarker, and the amount of analyte measured from the sample is diagnostic of a disease or medical condition. You can

  In any of the embodiments, the devices, systems, and methods of the present invention receive a report indicating a measure of biomarker and a range of biomarker measurements in individuals who are at or low risk of suffering from a disease or condition. Or further providing to a subject, the measured amount of biomarker over a range of measured values is a diagnosis of a disease or condition.

  In any of the embodiments, the devices, systems, and methods of the invention further comprise diagnosing the subject based on information that includes a measured amount of biomarker in the sample. In some cases, the diagnosing step includes transmitting data that includes the biomarker metric to a remote location and receiving a diagnosis from the remote location based on the information that includes the metric.

  In any embodiment, the biomarker may be selected from Table B1, B2, B3, or B7. In some examples, the biomarker is a protein selected from Table B1, B2 or B3. In some examples, the biomarker is a nucleic acid selected from Table B2, B3 or B7. In some examples, the biomarker is a biomarker from an infectious agent selected from Table B2. In some examples, the biomarker is a microRNA (miRNA) selected from Table B7.

  In an optional embodiment, the applying step b) isolates the miRNA from the sample to produce an isolated miRNA sample and treats the isolated miRNA sample with a disk coupled dot-on-pillar antenna (CROF). Device) including applying to an array.

  In any of the embodiments, the CROF device comprises multiple capture agents that bind to a biomarker selected from Tables 1, 2, 3, and/or 7, and the reading step d) can diagnose a disease or condition. Includes obtaining a measured amount of multiple biomarkers in the sample.

  In any embodiment, the capture agent can be an antibody epitope and the biomarker can be an antibody that binds to the antibody epitope. In some embodiments, the antibody epitope comprises a biomolecule selected from Table B4, B5, or B6 or a fragment thereof. In some embodiments, the antibody epitope comprises an allergen selected from Table 5 or a fragment thereof. In some embodiments, the antibody epitope comprises a biomolecule or fragment thereof from an infectious agent selected from Table 6.

  In any embodiments, the CROF device comprises a plurality of antibody epitopes selected from Tables B4, B5, and/or B6, and the reading step d) comprises a plurality of epitope-binding antibodies in a sample that can diagnose a disease or condition. Including obtaining the measured amount of.

  In any embodiment, the sample may be an environmental sample and the analyte may be an environmental marker. In some embodiments, environmental markers are selected from Table B8.

  In any embodiment, the method comprises receiving or providing a report indicating the safety or harm of a subject exposed to the environment in which the sample was obtained.

  In any of the embodiments, the method transmits data including a measured amount of environmental markers to a remote location and receives a report indicating the safety or harm of a subject exposed to the environment in which the sample was obtained. Including that.

  In any embodiments, the CROF device comprises a plurality of capture agents that bind to environmental markers selected from Table B8 and the reading step d) comprises obtaining a measured amount of a plurality of environmental markers in the sample. ..

  In any embodiment, the sample may be a food sample, the analyte may be a food marker, and the amount of food marker in the sample correlates with food safety for consumption. In some embodiments, the food marker is selected from Table B9.

  In any embodiment, the method comprises receiving or providing a report indicating the safety or harm of a subject consuming the food from which the sample was obtained.

  In any of the embodiments, the method comprises transmitting data including a measured amount of food markers to a remote location and receiving a report indicating the safety or harm of a subject consuming the food from which the sample was obtained. Including and

  In any of the embodiments, the CROF device array comprises a plurality of capture agents that bind to a food marker selected from Table B9, and obtaining comprises obtaining a measured amount of the plurality of food markers in the sample. Measured quantities of multiple food markers in correlate with food safety for consumption.

  The specification further provides kits for practicing the devices, systems and methods of the invention.

  Any volume of sample can be applied to the CROF device. Examples of volumes are about 10 mL or less, 5 mL or less, 3 mL or less, 1 microliter (μL, also referred to as “uL”) or less, 500 μL or less, 300 μL or less, 250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less, 125 μL Hereinafter, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less, 0.001 μL Hereinafter, examples include, but are not limited to, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1 pL or less, or a range between any two of these values.

  In a preferred embodiment, the volume of the sample applied to the CROF device and their fluctuation values are about 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 1 μL. Hereinafter, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less, 0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1 pL or less, or a range between any two of these values. But is not limited to.

  In another preferred embodiment, the volume of the sample applied to the CROF device and their variation values are about 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less, 0.5 μL or less, 0.1 μL or less, 0.05 μL or less. , 0.001 μL or less, 0.0005 μL or less, 0.0001 μL or less, 10 pL or less, 1 pL or less, or a range between any two of these values, but is not limited thereto.

  The sample volume may be about 1 drop of sample. The amount of sample may be the amount taken from the pricked finger or fingertip. The amount of sample may be the amount taken from a microneedle or intravenous draw.

  Once obtained from the sample source, the sample may be used without further processing or may be processed to concentrate the analytes of interest, remove large particulates, dissolve or resuspend solid samples, etc. May be achieved.

  The sample can be applied to the CROF device in any suitable manner. Suitable methods include using pipettes, droppers, syringes and the like. In certain embodiments, when the CROF device is placed on a dipstick type support, applying the sample to the CROF device by immersing the sample receiving area of the dipstick in the sample, as described below. You can

  The sample may be taken once or multiple times. Samples taken over time may also be aggregated and/or processed individually (by applying to a CROF device and obtaining a measured amount of analyte in the sample, as described herein). Good. In some instances, the measurement results obtained over time may be aggregated and may be used over time for longitudinal analysis to facilitate screening, diagnosis, treatment, and/or disease prevention. Good.

  As mentioned above, the CROF device can be washed in any convenient manner to remove unbound sample components. In certain embodiments, the binding buffer is used to wash the surface of the CROF device to remove unbound sample components.

  Detectable labeling of the analyte can be accomplished in any convenient way. The analyte may be labeled directly or indirectly. In direct labeling, the analyte in the sample is labeled before applying the sample to the CROF instrument. In indirect labeling, after applying the sample to the CROF instrument, as described below, the analyte in the unlabeled sample is labeled to obtain the unlabeled analyte.

Data Processing In certain embodiments, the apparatus of the present invention is configured to read a CROF device and process the acquired data. The device may be configured in any suitable manner to process the data in the method of the invention. In a particular embodiment, the device comprises a memory location for storage of instructions for data storage and/or data processing and/or for database storage. The data can be stored in memory in any suitable format.

  In a particular embodiment, the device comprises a processor for processing the data. In particular embodiments, the instructions for processing the data may be stored in the processor or may be stored in a separate memory location. In some embodiments, the device can include software for implementing the process.

  In a particular embodiment, a device configured to process data obtained from a CROF device includes a software implemented method of performing the process. Software implementation methods include image acquisition algorithms, image processing algorithms, user interface methods that facilitate interactions between users and computing devices, and act as means for data collection, transmission and analysis, communication protocols, and data processing. It may include one or more of the algorithms. In a particular embodiment, the image processing algorithm is configured for particle count, LUT (look-up table) filter, particle filter, pattern recognition, morphological determination, histogram, line profile, terrain representation, binary conversion, or color matching profile. Including one or more of them.

  In certain embodiments, the device is configured to display information on a video display or a touch screen display when the display page is interpreted by software resident in the device's memory. The display pages described herein are, for example, hypertext markup language ("HTML"), dynamic hypertext markup language ("DHTML"), extensible hypertext markup language ("XHTML"), extensible. Any suitable software language, such as Markup Language (“XML”), or another software language that may be used to create computer files that can be displayed on a video or other display in a manner perceptible by a user. Can be created using. Any software readable medium having logic, code, data, instructions may be used to implement any software or step or method. If the network includes the Internet, the display page may include any suitable type of web page.

  The display page according to the present invention may be, for example, a VBSscript routine, a JScript routine, a Javascript routine, a Java applet, an ActiveX component, an ASP. It may include built-in functionality including software programs stored in a memory device such as NET, AJAX, Flash applets, Silverlight applets, or AIR routines.

  The display page includes well-known features of graphical user interface technologies such as frames, windows, scroll bars, buttons, icons, and hyperlinks, and well-known features such as "click" or touch screen interfaces. Pointing and clicking on a graphical user interface button, icon, menu option, or hyperlink is also known as "selecting" a button, option, or hyperlink. The display page according to the present invention can incorporate multimedia features, multi-touch, pixel sensing, IR LED-based surfaces, vision-based interaction with or without a camera.

  The user interface may be displayed on a video display and/or display page. The user interface can display a report generated based on the analytical data for the sample, as described further below.

  The processor may be configured to process the data used in the method of the present invention in any suitable manner. The data is processed into, for example, binned data, transformed data (eg, time domain data transformed into the frequency domain by a Fourier transform), or in combination with other data. The processing can put the data into a desired format and can include changing the format of the data. The processing may include detection of signals from a sample, correction of raw data based on mathematical manipulations or corrections and/or calibrations specific to the device or reagents used to test the sample, calculation of values such as concentration values, ( Comparison with baselines, thresholds, standard curves, historical data or data from other sensors, etc., determination of whether the test is accurate, above or below normal or acceptable ranges, or abnormal conditions Highlighting of values or results that are outliers or that may cause concern, or a combination of results that can also indicate the presence of an abnormal condition, curve fitting, mathematical or other analytical inference ( Use of the data as a basis for (including deductive, inductive, Bayesian, or other inference), and other suitable forms of processing. In certain embodiments, the processing can include comparing the processed data to a database stored on the device to retrieve instructions for a sequence of actions performed by the subject.

  In certain embodiments, the device is configured to process the input data by comparing the input data with a database stored in memory to retrieve instructions for a sequence of actions performed by the subject. Good. In some embodiments, the database can include stored information, including thresholds for analytes of interest. The threshold may be used to determine the presence or concentration of one or more analytes. Thresholds may be used to detect situations where alerts are useful. The data storage unit may include records or other information useful in generating a report on the sample.

  In certain embodiments, the device may be configured to receive data from the CROF device. Thus, in some cases, the device may be configured to receive data not related to the sample provided by the subject, but relevant to the diagnosis. These data include, but are not limited to, age, sex, height, weight, individual and/or family medical history, and the like. In certain embodiments, the device is configured to process data derived from or independent of the sample applied to the CROF device.

Network In certain embodiments, the device is a network such as a local area network (LAN), a wide area network (WAN) such as the Internet, a personal area network, a telecommunication network such as a telephone network, a mobile phone network, a mobile network, a wireless network. , A data providing network, or any other type of network. In particular embodiments, the device may be configured to utilize a wireless technology such as Bluetooth or RTM technology. In some embodiments, the device is configured to utilize various communication methods such as dial-up wired connection using a modem, TI, integrated services digital network (ISDN) or direct link such as a cable line. Good. In some embodiments, the wireless connection is an exemplary wireless network such as a cellular, satellite, or pager network, (General Packet Radio Service) GPRS, or a local data transfer system such as Ethernet or Token Ring on a LAN. Can be used. In some embodiments, the device is capable of wireless communication using infrared communication components.

  In a particular embodiment, the device is configured to receive a computer file transmitted from a server over a network and stored in memory. A device may receive a tangible computer-readable medium, which may include instructions, logic, data, or code stored in permanent or temporary memory of the device, or affect operation by the device. May be initiated or an action may be initiated. One or more devices can communicate with computer files or links that can provide access to other computer files.

  In some embodiments, the device is a personal computer, server, laptop computer, mobile device, tablet, cell phone, mobile phone, satellite phone, smartphone (eg, iPhone, Android, Blackberry, Palm, Symbian, Windows), A personal digital assistant, a Bluetooth device, a pager, a landline phone, or other network device. Such a device may be a device capable of communicating. As used herein, the term "mobile phone" uses a telephone handset that can operate in a cellular network, a Voice over IP (VoIP) network such as Session Initiation Protocol (SIP), or an 802.11x protocol. Wireless local area network (WLAN), or any combination thereof. In certain embodiments, the device is handheld and compact so that it can fit into a consumer's wallet and/or pocket (eg, pocket size).

Microfluidic Channels In certain embodiments, the CROF device is an integrated microfluidic platform or microfluidic device. The microfluidic device is configured to receive the sample, detect the analyte in the sample using the CROF device, and have different regions for collecting the waste in the reservoir. Thus, in certain embodiments, as described above, the microfluidic channel platform guides a sample applied to the sample receiving area of the microfluidic device to a CROF device configured to detect an analyte. It can include components. The fluid handling component may be configured to guide one or more fluids to the microfluidic device. In some examples, the fluid handling components are configured to guide fluids such as, but not limited to, sample solutions, buffers, etc. Fluid handling components include, but are not limited to, passive pumps and microfluidic channels. In some cases, passive pumps are configured for capillary action driven microfluidic handling and fluid routing through the microfluidic devices disclosed herein. In particular examples, the microfluidic fluid handling component delivers a small amount of fluid, such as 100 μL or less, 50 μL or less, or 25 μL or less, or 10 μL or less, or 5 μL or less, or 500 μL or less, including 1 μL or less, such as 1 mL or less. Is configured as follows. Therefore, in certain embodiments, no external power source is required to operate the microfluidic device and to carry out the devices, systems and methods of the present invention.

  In certain embodiments, the microfluidic device has dimensions within the range of 5 mm x 5 mm to 100 mm x 100 mm, including dimensions of 50 mm x 50 mm or less, such as 25 mm x 25 mm or less, or 10 mm x 10 mm or less. In certain embodiments, the microfluidic device has a thickness in the range of 2 mm to 0.3 mm, or 5 mm to 0.1 mm, including 1 mm to 0.4 mm, such as 3 mm to 0.2 mm.

  In certain embodiments, the CROF device is placed in a container, eg, a well of a multi-well plate. The CROF device may also be integrated into the bottom or wall of the wells of a multiwell plate.

  In some embodiments, a support that includes a CROF device, such as a microfluidic device or a multi-well plate, can have a CROF device identifier included in the support. The identifier may be a physical object formed on a support such as a microfluidic device. In some cases, as described above, the identifier may be read by a handheld device such as a mobile phone or smartphone. In some embodiments, a camera can capture an image of the identifier and analyze the image to identify a CROF device included in the microfluidic device. In one example, the identifier may be a barcode. The barcode may be a 1D or 2D barcode. In some embodiments, the identifier can emit one or more signals that can identify the signal enhancement detector. For example, the identifier can provide infrared, ultrasound, light, voice, electrical or other signals that can indicate the identity of the CROF device. The identifier may utilize a radio frequency identification (RFID) tag.

  The identifier can include information that enables the determination of the particular type of CROF device present in the microfluidic device or multiwell plate. In certain embodiments, the identifier provides a key to the database that associates each identifier key with unique information about the type of CROF device present in the microfluidic device or multiwell plate. Information specific to the type of CROF device includes the identification of the analytes that the CROF device is configured to detect, the coordinates of the position where a particular analyte can bind to the CROF device, the detection sensitivity for each analyte. Including, but not limited to. The database may include other information associated with a particular CROF device, including expiration date, lot number, etc. The database may reside on the handheld device and be provided on a computer-readable medium, or it may reside on a remote server accessible by the handheld device.

  A further aspect of the method of the invention comprises providing or receiving a report indicating the measured amount of the analyte and other information related to the source from which the analyte was obtained, eg diagnostic or health status of a diagnostic sample. , Exposure risk of environmental samples, health risk of food samples, etc. Reports include viewing reports displayed on the screen of the device, viewing email or text messages sent to the subject, listening to voice messages generated by the device, sensing vibrations generated by the device. It may be provided and received in any convenient format, including, but not limited to.

  The report may include any suitable information related to the source from which the analyte was obtained. In some examples, the report includes light data, including light intensity, wavelength, polarization, as well as other data about the light, such as absorbance data, transmission data, turbidity data, luminosity data, wavelength data (one , Including intensity at two or more wavelengths or a range of wavelengths), reflectance data, refractive index data, birefringence data, polarization and other optical data, photomultiplier tube, photodiode, charge coupled Output from photodetectors, such as devices, photometers, spectrophotometers, cameras, and other photodetection components and devices; image data, eg, data from digital cameras; used to obtain data Identifier information related to the CROF device, which may include data processed as described above. The report can represent a qualitative or quantitative aspect of the sample.

  In certain embodiments, the report reports to the subject the presence or absence of the analyte, the concentration of the analyte, the presence or absence of a secondary medical condition known to be associated with the presence or level of the analyte, the presence or level of the analyte. Probability or likelihood of a secondary medical condition known to be associated, likelihood of developing a secondary medical condition known to be associated with presence or level of analyte, presence of analyte or May indicate a change in the likelihood of developing a secondary medical condition known to be associated with a level, the progression of a secondary medical condition known to be associated with the presence or level of the analyte, etc. it can. Secondary medical conditions known to be associated with the presence or level of an analyte include the disease or condition of the diagnostic sample, the toxicity or other harmful environment of the environmental sample, spoilage or contamination of food samples, etc. But it's okay. In certain embodiments, the report includes instructions prompting or recommending the user to take action, which may include, for example, seeking medical assistance, dosing, stopping, and starting movement. Etc. The report may include alerts. One example of an alert is if an error is detected in the device or if the analyte concentration exceeds a predetermined threshold. The content of the report is presented in any suitable format, including text, graphs, graphics, animations, sound, voice, and vibration.

  In a particular embodiment, the report provides action advice to the user of a device of the invention, eg a mobile phone. This advice is given according to the test data by the device (eg detector+cell phone) together with one or more data sets and includes the date preloaded on the mobile device, the data on the accessible storage device, Without limitation, the storage device may be locally available or remotely accessible.

  In a particular embodiment, according to the scheme, each of the above advices has its own color on the display of the mobile phone.

  In certain embodiments, the devices, systems, and methods of the present invention include transmitting data including a measured amount of an analyte to a remote location and receiving an analysis from the remote location, such as diagnostics, safety information, or the like. . As mentioned above, sending data to a remote location can be accomplished in any convenient manner. Such transmission may be via electronic signals, radio frequency signals, optical signals, cellular signals, or any other type of signal transmitted via a wired or wireless connection. The transmission of any data, the description of electronic data, the transmissions described elsewhere herein include electronic signals, radio frequency signals, optical signals, cellular signals, or otherwise transmitted via a wired or wireless connection. Via any type of signal. The transmitted data may include data obtained from the CROF device and/or processed data and/or generated reports. The transmitted data further does not directly represent data that is not obtained from the CROF device, ie one aspect of the sample obtained from the subject, but as described above, other aspects of the subject from which the sample was obtained. It may include data to represent.

  A further aspect of the present disclosure includes a CROF device that includes multiple capture agents, each of which binds to multiple analytes in a sample, ie a multiplexed CROF device. In such cases, a CROF device containing multiple capture agents is configured to detect different types of analytes (proteins, nucleic acids, antibodies, etc.). Different analytes can be distinguished from one another in the array based on their position in the array, the emission wavelength of the detectable label that binds to the different analytes, or a combination of the above.

  In certain embodiments, the devices, systems, and methods of the present invention apply a control sample containing a known detectable amount of analyte to a control CROF device that includes a capture agent that binds to the analyte. Reading a control CROF instrument to obtain a control measurement of a known detectable amount of analyte in the sample. In certain embodiments, when the CROF device is in a microfluidic device, the control CROF device may be in the same device as the CROF device to which the test sample has been applied. In certain embodiments, control measurements obtained from a control sample can be used to obtain the absolute amount of analyte in the test sample. In certain embodiments, the control measurement results obtained from the control sample can be used to obtain a normalized relative amount of analyte in the test sample.

Utility The methods of the invention find use in a variety of different applications where it is desired to determine and/or quantify the presence or absence of one or more analytes in a sample. For example, the method of the present invention is used to detect proteins, peptides, nucleic acids, synthetic compounds, inorganic compounds and the like.

  In certain embodiments, the methods of the invention are used to detect nucleic acids, proteins, or other biomolecules in a sample. The method can include the detection of a set of biomarkers in the sample, eg, two or more distinct protein or nucleic acid biomarkers. For example, the method may be used for rapid clinical detection of two or more disease biomarkers in a biological sample, eg, for diagnosing a disease state in a subject, or for ongoing management or treatment of a disease state in a subject. Etc. may be used. As noted above, communication with a physician or other healthcare provider will ensure that the physician or other healthcare provider is aware and aware of potential concerns, and thus may take appropriate action. Sex can be higher.

  Applications of the devices, systems and methods of the present invention using a CROF device include (a) infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric disorders, neuropsychiatric disorders, and organic disorders such as lung disorders. Detection, purification and quantification of compounds or biomolecules that correlate with a specific disease stage such as kidney disease, (b) environment such as water and soil, or virus, fungus and bacterium from biological samples such as tissues and body fluids Detection, purification and quantification of microorganisms of (c), (c) Hazardous waste that poses a risk to food safety or national security, detection and quantification of chemical substances or biological samples such as anthrax, (d) glucose, blood oxygen Quantification of important parameters in medical or physiological monitors such as level and complete blood count, (e) detection and quantification of specific DNA or RNA from biological samples such as cells, viruses and body fluids, (f) genomic analysis Sequencing and comparison of gene sequences in DNA in chromosomes and mitochondria for, or (g) detection of reaction products during, for example, synthesis or purification of pharmaceuticals, but is not limited thereto. Some specific applications of the device, system and method of the present invention are described in more detail below.

Diagnostic Methods In certain embodiments, the methods of the invention are used to detect biomarkers. In some embodiments, the devices, systems, and methods of using the CROFs of the present invention may include the presence or absence of specific biomarkers and blood, plasma, serum, or, but not limited to, urine, blood, serum, plasma, saliva. Specific in other body fluids or excretions such as, semen, prostatic fluid, nipple aspirate, tear fluid, sweat, feces, buccal swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue It may be used to detect an increase or decrease in biomarker concentration. Thus, samples, eg diagnostic samples, may include different types of liquid or solid samples.

  In some examples, the sample may be a body fluid sample from the subject to be diagnosed. In some examples, a solid or semi-solid sample may be provided. These samples may include tissues and/or cells taken from the subject. The sample may be a biological sample. Examples of biological samples are blood, serum, plasma, nasal swabs, nasopharyngeal lavage fluid, saliva, urine, gastric juice, spinal fluid, tears, stool, mucus, sweat, earwax, oil, glandular secretions, cerebrospinal fluid. , Tissue, semen, vaginal fluid, tumor tissue-derived interstitial fluid, eye fluid, spinal fluid, pharyngeal swab, exhalation, hair, fingernails, skin, biopsy, placental fluid, amniotic fluid, cord blood, lymph fluid, cavity fluid , But not limited to, sputum, pus, microbiota, meconium, breast milk, exhaled breath condensate, and/or other excretions. The sample may include a nasopharyngeal wash. Nasal swabs, pharyngeal swabs, stool samples, hair, fingernails, ear lobes, exhaled breath, and other solid, semi-solid, or liquid samples, for example, are treated in extraction buffer before analysis for a fixed or variable amount of time. You may. In some cases, the extraction buffer or an aliquot thereof may then be treated like any other liquid sample. Examples of tissue samples of a subject include, but are not limited to, tightened tissue, muscle tissue, nerve tissue, epithelial tissue, cartilage, cancerous samples or bone.

  In some examples, the subject for whom the diagnostic sample is obtained may be a healthy individual, or at least an individual suspected of having a disease or health condition. In some examples, the subject may be a patient.

  In certain embodiments, the CROF device comprises a capture agent that is configured to specifically bind a biomarker in a sample provided by the subject. In particular embodiments, the biomarker may be a protein. In certain embodiments, the biomarker protein is specifically bound by the antibody capture agent present in the CROF device. In certain embodiments, the biomarker is an antibody that is specifically bound by the antigen capture agent present in the CROF device. In certain embodiments, the biomarker is a nucleic acid that is specifically bound by a nucleic acid capture agent, the nucleic acid capture agent is complementary to one or both of the double-stranded nucleic acid biomarkers, or Is complementary to the chain biomarker. In certain embodiments, a biomarker is a nucleic acid that is specifically bound by a nucleic acid binding protein. In certain embodiments, biomarker proteins are specifically bound by aptamers.

  The presence or absence of a biomarker or a significant change in biomarker concentration may be used to diagnose disease risk, diagnose the presence of disease in an individual, or adjust treatment of a disease in an individual. For example, the presence of a particular biomarker or group of biomarkers influences the choice of drug treatment or regimen given to an individual. When assessing potential drug therapies, biomarkers can be used as alternatives to natural endpoints such as survival or irreversible disease. If the treatment modifies a biomarker that is directly related to improved health, the biomarker can be used as an alternative endpoint to assess the clinical benefit of a particular treatment or dosing regimen. Thus, personalized diagnosis and treatment based on a particular biomarker or panel of biomarkers detected in an individual is facilitated by the methods of the invention. Moreover, as mentioned above, early detection of disease-related biomarkers is facilitated by the high sensitivity of the devices, systems and methods of the invention. Due to the ability to detect multiple biomarkers on mobile devices such as smartphones that combine sensitivity, scalability and ease of use, the methods of the present disclosure are used for portable and point-of-care or bedside molecular diagnostics.

  In certain embodiments, the methods of the invention are used to detect biomarkers for diseases or disease states. In some cases, the methods of the invention are used to characterize cell signaling pathways and detect biomarkers for intracellular communication for drug discovery and vaccine development. For example, the methods of the invention may be used to detect and/or quantify the amount of biomarkers in a pathological, healthy or benign sample. In certain embodiments, the methods of the invention are used to detect biomarkers of infectious diseases or disease states. In some cases, the biomarker may be a molecular biomarker, including but not limited to proteins, nucleic acids, carbohydrates, small molecules and the like.

  The methods of the present invention include, but are not limited to, detection and/or quantification of biomarkers as described above, screening assays that test samples at regular intervals for asymptomatic subjects, presence and/or amount of biomarkers. Used in diagnostic assays such as prognostic assays to predict possible disease course, stratified assays that can predict subject response to different drug treatments, and efficacy assays where drug efficacy is monitored .

  In some embodiments, the subject biosensor can be used to diagnose a pathogen infection by detecting a target nucleic acid from the pathogen in a sample. Target nucleic acids include, for example, human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia virus and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus. Virus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papilloma virus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus. 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma herpesvirus), and yellow fever virus, Japanese encephalitis virus, West Nile virus, and Ebola virus It may be from a virus selected from the group consisting of flaviviruses. However, the invention is not limited to the detection of nucleic acids, such as DNA or RNA, sequences of the above viruses, and can be successfully applied to other important pathogens in veterinary and/or human medicine.

  Due to DNA sequence homology, human papillomavirus (HPV) is further subdivided into over 70 different types. These types cause various diseases. HPV types 1, 2, 3, 4, 7, 10 and 26-29 cause benign warts. 5, 8, 9, 12, 14, 15, 17, 19-25, and 46-50 HPV cause lesions in patients with compromised immune systems. Types 6, 11, 34, 39, 41-44, and 51-55 cause benign pointed warts on the genital area and airway mucosa. Type 16 and 18 HPV cause epithelial dysplasia of the reproductive mucosa and are highly associated with invasive cancers of the cervix, vagina, vulva, and anal canal, thus making it of special medical interest. It is a thing. Human papillomavirus DNA integration is believed to be crucial in carcinogenesis of cervical cancer. Human papillomavirus can be detected, for example, from the DNA sequences of its capsid proteins L1 and L2. Therefore, the method of the invention applies in particular to detecting the DNA sequences of HPV 16 and/or 18 in tissue samples in order to assess the risk of developing cancer.

  Other pathogens that can be detected in diagnostic samples using the devices, systems, and methods of the invention include, but are not limited to: Varicella zoster, Staphylococcus epidermidis, Escherichia coli, methicillin. Resistant Staphylococcus aureus (MSRA), Staphylococcus aureus, Staphylococcus hominis, Fecal Enterococcus faecalis, Pseudomonas aeruginosa, Staphylococcus Capitis (Staphylococcus capitis), Staphylococcus warneri, Klebsiella pneumoniae, Haemophilus influenzae, Staphylococcus simulans p. -Albicans (Candida albicans), gonorrhea (Neisseria gorrhoeae), syphilis (Treponena pallidum), clamydia (Clamyda tracomitis), non-gonococcal urethraemia (ureaplasma) (Ureaplasm urealyticum)), chancroid (Haemophilus ducrey), trichomoniasis (Trichomonas vaginalis), Pseudomonas aeruginosa, methicillin-resistant Staphylococcus aureus (MSRA), Klebsiella pneumoniae, Haemophilus influenzae, Yellow grape Coccus, Stenotrophomonas maltophilia, Haemophilis parainfluenzae, Escherichia coli, faecal streptococci, Serratia marcescens, Haemophilus parahaemolyticus (Haemophilis p) arahaemolyticus), Enterococcus cloacae, Candida albicans, Moraxella catarrhalis, Streptococcus pneumoniae, Citrobacter freundii, Enterococcus faecium, Enterococcus faecium. -Oxytoca, Pseudomonas fluorscens, Neiseria meningitidis, Streptococcus pyogenes, Pneumocystis carinii, Pneumocystis carinii, Klebsiella pneumoniae -Pneumophila (Legionella pneumophila), Mycoplasma pneumoniae (Mycoplasma pneumoniae), Mycobacterium tuberculosis, etc., and those listed in Tables B2 and B6.

  In some cases, the CROF instrument can be used to detect biomarkers that are present in low concentrations. For example, a CROF device detects cancer antigens in easily obtained body fluids (eg, blood, saliva, urine, tears, etc.) and easily obtains body fluids (eg, neuropathy (eg, Alzheimer's antigen)). Biomarkers) for the detection of tissue-specific disease biomarkers, for detecting infections (especially for detection of low titer latent viruses such as HIV), and for detecting fetal antigens in maternal blood, and for example in subjects. It may be used for the detection of exogenous compounds (eg drugs or pollutants) in the bloodstream of.

  Tables B1-B3 below provide a list of biomarkers and their associated diseases that can be detected using the CROF device of the present invention (and when used in combination with a suitable monoclonal antibody, nucleic acid or other capture agent). One of the potential sources of biomarkers (eg "CSF", cerebrospinal fluid) is also shown in the table. In many cases, the biosensor of the present invention can detect biomarkers in different displayed body fluids. For example, biomarkers found in cerebrospinal fluid (CSF) can be identified in urine, blood, or saliva. It will be apparent to those skilled in the art that the CROF device of the present invention can be used to capture and detect more biomarkers known in the art that contribute to disease or health condition diagnosis.

  Unless otherwise specified, a biomarker may be a protein or nucleic acid (eg mRNA) biomarker. Unless otherwise specified, diagnosis may be associated with increased or decreased levels of biomarkers in a sample. Tables 1 and 2 of US Provisional Patent Application No. 62/234,538 filed on September 29, 2015 show biomarkers, diseases that can be diagnosed by the biomarkers, and samples in which the biomarkers can be detected. Is set forth herein, which application is incorporated herein by reference.

  In some examples, the devices, systems, and methods of the invention are used to inform a sample source subject of their health status. Health conditions diagnosed or measured by the device, system, and method of the present invention include chemical balance, nutritional health, exercise, fatigue, sleep, stress, prediabetes, allergy, aging, environmentally harmful substances, and pesticides. Including but not limited to herbicides, exposure to synthetic hormone analogs, pregnancy, menopause and menopause. Table 3 of US Provisional Application No. 62/234,538, filed September 29, 2015, shows that the present CROF device (when used in combination with a suitable monoclonal antibody, nucleic acid, or other capture agent). ) Provides a list of biomarkers and their associated health conditions that can be detected with the application of which is incorporated herein by reference.

  In some examples, the biomarker that can be detected by the devices, systems, and methods of the invention is an antibody in the sample, e.g., a diagnostic sample, which diagnoses a disease or condition in a subject from which the sample is derived. You can The CROF device for detecting antibody analytes may include an antibody epitope, to which the antibody analyte is specifically bound as a capture agent. In some cases, the disease or condition is associated with an autoimmune disease and induces an autoimmune response against its own antibody (autoantibodies). In some embodiments, the antibody analyte of interest is the antibody IgA, IgM, IgE, IgD, or IgG antibody. In some examples, the labeling agent comprises a moiety that is specifically bound to a region of the antibody analyte, the antibody analyte having specificity for a particular type of antibody. For example, a labeling agent comprising peptide M, SSL7 or Jacalin can specifically bind IgA, and a labeling agent comprising protein G can specifically bind IgG. Protein L is used to bind all types of antibodies.

  Table B4 provides a list of autoantibody targets, used in whole or as epitope fragments as capture agents in the devices, systems, and methods of the invention to quantify the amount of epitope-binding antibody analytes in a sample. It can be measured and thus diagnosed a related disease or condition, eg an autoimmune disease. In some cases, the disease or condition is associated with an immune response to the allergen. Table B5 provides a list of antigenic species, used in their entirety or as epitope fragments, as capture agents in the devices, systems, and methods of the invention to quantify the amount of epitope-binding antibody analytes in a sample. Therefore, related diseases or conditions, such as allergies, can be diagnosed. In a particular example, the disease or condition is associated with an infectious disease, and the pathogen is based on information that includes a measured amount of antibodies to one or more epitopes from the pathogen (eg, lipopolysaccharide, toxin, protein, etc.). Can be diagnosed. Table B6 provides a list of epitopes derived from pathogens, which, either in whole or as an epitope fragment, is used as a capture agent in the devices, systems, and methods of the present invention to detect epitope-binding antibody analytes in a sample. It can be quantified and thus diagnosed for a related disease or condition, such as an infection. Other epitopes or antigens suitable for use in the present diagnostic method are, for example, as described in PCT application WO 2013164476, which is incorporated herein by reference. It will be apparent to those skilled in the art that the CROF device of the present invention can be used to capture and detect more antibody analytes that contribute to the diagnosis of a disease or condition. By arranging the CROF device, the epitopes present in the CROF device are not cross-reactive, ie, bound by antibodies that non-specifically bind to many epitopes present in the CROF device.

  Table 4-6 of US Provisional Patent Application No. 62/234,538, filed September 29, 2015, shows typical autoantibody epitopes, allergen epitopes, and pathogens that can be detected using this method. A list of epitopes (each) is described, which application is incorporated herein by reference.

  In some examples, the biomarker detected by the devices, systems and methods of this invention is a microRNA (miRNA) biomarker, which is associated with a disease or condition. Table B7 below provides a list of miRNA biomarkers and their associated diseases/conditions that can be detected using the present CROF device (when used in combination with a suitable complementary nucleic acid or other capture agent). Table 7 of U.S. Provisional Patent Application No. 62/234,538, filed September 29, 2015, illustrates a list of exemplary miRNA biomarkers and diseases that can be diagnosed using the miRNAs, and said application. Are incorporated herein by reference.

  The method of the invention is further used in validation assays. For example, validation assays can be used to validate or confirm that potential disease biomarkers are a reliable indicator of the presence or absence of disease across many individuals. The assay time of the method is short, which can facilitate increased throughput for screening multiple samples in the shortest amount of time.

  In some examples, the method can be used without the need for a working laboratory setting. Compared to comparable analytical laboratory equipment, the method provides comparable analytical sensitivity in portable handheld systems. In some cases, weight and operating costs are lower than in typical fixed laboratory equipment. Further, the method can be used in a home environment for home testing without physician prescription by untrained persons to detect one or more analytes in a sample. The method may also be used for rapid diagnosis in a clinical environment, for example, in an environment in which a fixed research laboratory apparatus is not installed due to reasons such as the bedside of the sick or cost.

  As described above, the CROF device of the present invention is used to detect nucleic acid in a sample. The CROF device of the present invention may be used for various drug discovery and research applications in addition to the above-mentioned diagnostic applications. For example, the CROF device of the present invention has many applications, including the diagnosis or monitoring of diseases or conditions (the presence of nucleic acids provides biomarkers for the disease or conditions), discovery of drug targets (eg, Nucleic acids are differentially expressed in diseases or conditions and can be targeted for drug therapy), drug screening (the effect of the drug is monitored by assessing the level of the nucleic acid), drug sensitivity (drug sensitivity is Including the determination of the presence of nucleic acids in a sample, or the relative levels of particular nucleic acids in two or more samples in certain embodiments). , But not limited to them.

  In certain embodiments, relative levels of nucleic acids in two or more different nucleic acid samples may be obtained and compared using the methods described above. In these embodiments, the results obtained from the above methods are typically normalized and compared to the total amount of nucleic acid (eg, constitutive RNA) in the sample. This may be done by comparison to a ratio or other means. In certain embodiments, the nucleic acid profiles of two or more different samples can be compared to identify nucleic acids associated with a particular disease or condition.

  In some examples, the different samples may be composed of an "experimental" sample, that is, a sample of interest and a "control" sample that is comparable to the experimental sample. In many embodiments, the different samples are pairs of cell types or fragments thereof, one cell type being the cell type of interest, for example abnormal cells, and the other cell type being a control cell type, For example, normal cells. When comparing two fragments of a cell, the fragment is usually the same fragment from each of the two cells. However, in certain embodiments, two fragments of the same cell can be compared. Exemplary cell type pairs are, for example, tissue biopsies, usually from the same patient (eg, colon, breast, prostate, lung, tissue with a disease such as skin cancer, or tissue infected with a pathogen, etc.). Cells isolated from and from normal cells from the same tissue and infected or treated by pathogens (eg, peptides, hormones, altered temperature, growth conditions, physical stress, cell transformation, etc. Environmental or chemical factors) Cells grown in immortal tissue culture (eg, cells with a proliferative mutation or immortalizing transgene) and normal cells (eg, not immortal, uninfected or treated). A cell that is identical to the experimental cell, except that it is not present), and a mammal that has been exposed to cancer, disease, an aged mammal, or a condition, and a mammal of the same species, preferably healthy or adolescent. Includes cells isolated from a mammal having cells of mammalian origin, a differentiated cell and an undifferentiated cell from the same mammal (eg, a precursor cell of another cell in the mammal) . In one embodiment, different types of cells may be used, eg, neuronal and non-neuronal cells, or cells in different states (eg, before and after stimulation on the cells). In another embodiment of the invention, the experimental material is a cell susceptible to infection by a pathogen, such as a virus such as human immunodeficiency virus (HIV), and the reference material is resistant to infection by the pathogen. Cells that have. In another embodiment of the invention, the sample pair is represented by undifferentiated cells and differentiated cells such as stem cells.

  As mentioned above, aspects of the method include providing or receiving a report indicating a measured amount of an analyte such as a biomarker in a sample. In some cases, the sample is a diagnostic sample and the report may further include a range of biomarker measurements in individuals who are at or low risk of suffering from a disease or condition, measurements obtained from healthy individuals. The measured amount of the biomarker in the diagnostic sample obtained from the subject for the range of is a diagnosis of a disease or pathological condition. In such an example, if the measured value of the biomarker in the sample provided by the subject is outside the expected range of the biomarker of a healthy individual, the subject is prone to develop the disease or condition or has the disease or condition. Has a higher chance of suffering. In some cases, the measured amount of biomarker and range of values obtained from healthy individuals is normalized to a given standard to allow comparison.

  In certain aspects, the report includes the presence or absence of the biomarker in the subject, the concentration of the biomarker, the presence or absence of the disease or condition, the probability or likelihood that the subject suffers from the disease or condition, the likelihood of developing the disease or condition, the disease or condition. May indicate a change in the possibility of developing the disease, the progress of a disease or a pathological condition, and the like. Diseases or conditions reported include cancer, inflammatory diseases such as arthritis, metabolic diseases such as diabetes, ischemic diseases such as stroke or heart attack, and neurodegeneration such as Alzheimer's disease or Parkinson's disease. Diseases, organ failure such as kidney or liver failure, overdose, stress, fatigue, muscle damage, and pregnancy-related symptoms such as non-invasive prenatal testing. . In certain embodiments, the report includes instructions prompting or recommending that the patient take action, which action may include, for example, seeking medical assistance, dosing, stopping, and initiating movement. Etc. The report may include alerts. One example of an alert is if an error is detected in the device or if the analyte concentration exceeds a predetermined threshold. The content of the report is presented in any suitable format, including text, graphs, graphics, animations, sound, voice, and vibration.

  In a particular embodiment, the report provides action advice to the user of a device of the invention, eg a mobile phone. This advice is given according to the test data by the device (eg detector+cell phone) together with one or more data sets and includes the date preloaded on the mobile device, the data on the accessible storage device, Without limitation, the storage device may be locally available or remotely accessible.

  Advice is (i) normal (good day), (ii) should be monitored frequently, (iii) the following parameters should be rigorously checked (and list the parameters) , (Iv) certain parameters of the subject are on the border and should be checked daily, (v) certain parameters are above the threshold and should be seen within a certain number of days, This includes, but is not limited to, either (vi) to be seen immediately and (vii) to go to the emergency room immediately.

  In some embodiments, if the device determines that the subject needs to visit or go to the emergency room, the device automatically sends such a request to the physician and the emergency room.

  In some embodiments, if the request automatically sent by the device is not answered by the doctor or the emergency room, the device repeatedly sends the request at predetermined time intervals.

  In certain embodiments, the report warns of possible conflicts between advice based on information obtained from samples provided by the subject and any contraindications based on the subject's health history or profile. May be provided.

  In certain embodiments, the method comprises diagnosing the subject based on information provided by the subject, including the measured amount of the biomarker in the sample. In addition to data regarding measured biomarkers in a sample (eg, biomarker type, amount of biomarker in the sample), information used to diagnose the subject may be the subject's age, gender, height, weight, or Other data related to the subject, including but not limited to personal and/or family medical history, may also be included.

  In some embodiments, the diagnosing step comprises transmitting data including a measured amount of biomarkers to a remote location and receiving the diagnosis from the remote location. Diagnosing a subject based on information including biomarkers detected by a CROF device may be accomplished by any suitable means. In certain embodiments, diagnosis is performed by a medical professional who may be located with the subject or may be located remotely. In another embodiment, the medical professional accesses the data transmitted by the device at a remote location or a third location different from the subject's location. Medical professionals may include individuals or organizations associated with a healthcare system. The health care professional may be a health care professional. The medical professional may be a doctor. A health care professional may be an individual or institution that systematically provides preventative, curative, facilitative, or rehabilitative health care services to individuals, families, and/or communities. Examples of medical professionals are doctors (including general practitioners and specialists), dentists, physician assistants, nurses, midwives, pharmacologists/pharmacists, dietitians, therapists, psychologists, shiatsu, clinical staff, physiotherapy. Trained to provide medical professionals, blood sampling specialists, occupational therapists, optometrists, emergency medical technicians, medical assistants, medical examination technicians, medical prosthesis technicians, x-ray technicians, social workers, and various healthcare services. It may include a wide variety of human resources. The medical professional may or may not be authorized to write a prescription. Medical professionals may work in or belong to hospitals, healthcare centers and other service delivery points, or academic training, research, and management. Some medical professionals may provide care and treatment services to patients in private homes. Community health workers may work outside formal health care institutions. Health care services, health record managers and health information technicians, and other support workers may be health care professionals or may belong to health care providers.

  In some embodiments, the medical professional may be familiar with or in contact with the subject. The subject may be a medical professional patient. In some examples, a medical professional may instruct a subject to undergo a clinical examination. In one example, the medical professional may be the subject's primary care physician. The medical professional may be any type of physician for the subject, including general practitioners and specialists.

  Thus, the medical professional may also analyze or review the reports generated by the device that captured the optical signal from the CROF device or the data transmitted from the device and/or the results of the analysis performed at the remote location. Good. In certain embodiments, the medical professional may send instructions or recommendations to the subject based on the data sent by the device and/or the data analyzed remotely.

Environmental Testing As summarized above, the devices, systems and methods of the present invention may be used to analyze the presence or absence of environmental markers in environmental samples, such as water, soil, industrial waste and the like. The environmental marker may be any suitable marker as shown in Table B8 below that can be captured by a capture agent that specifically binds to the environmental marker in a CROF device composed of a capture agent. Environmental samples may be obtained from any suitable source such as rivers, seas, lakes, rain, snow, sewage, wastewater treatment wastewater, agricultural wastewater, industrial wastewater, tap water, or drinking water. In some embodiments, the presence or level of quantitation of environmental markers in a sample may indicate the state of the environment in which the sample was obtained. In some cases, the environmental marker may be a substance that is toxic or harmful to humans, companion animals, plants, etc. exposed to the environment. In some cases, the environmental marker may be an allergen that may cause an allergic reaction in some individuals exposed to the environment. In some cases, the presence or level of quantitation of environmental markers in a sample may correlate with overall environmental health. In such cases, overall environmental health may be measured over a period such as weeks, months, years, or decades.

  In some embodiments, the devices, systems and methods of the present invention further include receiving or providing a report, wherein the report is based on information obtained based on information including a measured amount of environmental markers. Indicates the safety or harm of subjects exposed to. The information used to assess the environmental safety risk or health may include data other than the type and measure of environmental markers. These other data may include location, altitude, temperature, time of day/month/year, pressure, humidity, wind direction and speed, weather, etc. This data can be averages or trends over a given period of time (minutes, hours, days, weeks, months, years, etc.), or over shorter time periods (milliseconds, seconds, minutes, etc.). It may represent an instantaneous value.

  This report may be generated by a device configured to read the CROF device, or it may be generated at a remote location when transmitting data including measured quantities of environmental markers. In some cases, an expert may have access to data at or transmitted to a remote location and may analyze or review the data to generate a report. The expert may be a government agency such as the Centers for Disease Control and Prevention (CDC) or the US Environmental Protection Agency (EPA), a research institute such as a university, or a scientist or manager at a private company. In certain embodiments, the expert may send instructions or recommendations to the user based on the data sent by the device and/or the data analyzed remotely.

  Table 8 of U.S. Provisional Patent Application No. 62/234,538, filed September 29, 2015, illustrates a list of exemplary environmental markers, which application is incorporated herein by reference.

Food Testing As summarized above, the devices, systems and methods of the present invention are for analyzing the presence or absence of food markers in food samples, such as raw foods, processed foods, cooked foods, drinking water, and the like. May be used for. The food marker can be any suitable marker as shown in Table B9 below that can be captured by a capture agent that specifically binds to the food marker in a CROF device composed of a capture agent. Environmental samples may be obtained from any suitable source such as tap water, drinking water, prepared foods, prepared foods, or raw foods. In some embodiments, the presence or quantitative level of food markers in the sample may indicate safety or harm to the subject when the food is consumed. In some embodiments, the food marker is a substance from a pathogenic organism or microorganism that indicates the presence of the organism in the food from which the sample was obtained. In some embodiments, a food marker is a toxic or harmful substance when consumed by a subject. In some embodiments, the food marker may be a bioactive compound, which may unintentionally or unexpectedly alter physiology when consumed by a subject. In some embodiments, the food marker indicates how the food was obtained (cultivation, acquisition, harvesting, harvesting, processing, cooking, etc.). In some embodiments, the food marker indicates a nutritional component of the food. In some embodiments, the food marker is an allergen, which can induce an allergic reaction when the food from which the sample was obtained is consumed by the subject.

  In some embodiments, the devices, systems, and methods of the present invention further include receiving or providing a report, wherein the report was obtained based on information including a measured level of food markers. Indicates the safety or harm of a subject who consumes food. The information used to assess the safety of food for consumption may include data other than food marker type and measured quantity. These other data may include any health condition associated with the consumer (allergy, pregnancy, chronic or acute illness, current prescription drug, etc.).

  This report may be generated by a device configured to read the CROF device, or it may be generated at a remote location when transmitting data including a measure of a food marker. In some cases, a food safety professional may access data that is at or transmitted to a remote location and may analyze or review the data to generate a report. The food safety expert may be a government agency such as the US Food and Drug Administration (FDA) or the Centers for Disease Control and Prevention (CDC), a research institute such as a university, or a scientist or manager at a private company. In certain embodiments, the food safety professional may send instructions or recommendations to the user based on the data sent by the device and/or the data analyzed remotely.

  Table 8 of U.S. Provisional Patent Application No. 62/234,538, filed September 29, 2015, illustrates a list of exemplary food markers, which application is incorporated herein by reference.

Kits Aspects of the present disclosure include kits used to carry out the devices, systems, and methods of the invention described above. In certain embodiments, the kit comprises a CROF device configured to specifically bind to the analyte, eg, the analyte is in Table B1, B2, B3, B7, B8, B9 or Table B4, B5. , And antibody analytes that specifically bind to the epitopes listed in B6. In a particular embodiment, the kit includes instructions for performing the method with a handheld device such as a cell phone. These instructions may be present in the kit in various forms, one or more of which may be present in the kit. One form of these instructions for use is as information printed on a suitable medium or substrate, such as one or more sheets of printed information, on the packaging of the kit, package insert, etc. Good. Another means is a computer readable medium having information recorded or stored thereon, such as a floppy disk, CD, DVD, Blu-ray, computer readable memory or the like. Yet another means that may exist is the address of a website that may access information at the deleted site via the Internet. The kit may further include software for performing the method for measuring an analyte on a device provided on a computer-readable medium as described herein. Any convenient means may be present in the kit.

  In some embodiments, the kit comprises a detectable label, such as a fluorescently labeled antibody or oligonucleotide that specifically binds to the analyte of interest, for detection to label the analyte of interest. Including agents. The detection agent may be provided in a container separate from the CROF device, or may be provided in the CROF device.

  In some embodiments, the kit comprises a control sample containing a known detectable amount of analyte detected in the sample. The control sample may be provided in a container and may be present in solution at a known concentration, or may be provided in a dry form such as lyophilized or lyophilized. The kit may further include a buffer for use in dissolving the control sample when it is provided in dry form.

25 Blood Tests Some exemplary embodiments of the application of the present invention simply and quickly count blood cells using a smartphone.

  In some embodiments, the first plate and the second plate are selected from relatively flat surface thin glass slides (eg 0.2 mm thick) or thin plastic films (eg 15 mm thick). And each has a region having a length and width of about 0.5 cm to 10 cm. The spacers are made of glass, plastic, or other material that does not significantly deform under the press. Prior to sample attachment, spacers are placed on the first plate, the second plate, or both, and the blood count (staining dye and/or anticoagulant on the first plate, the second plate, or both). ) Promoting reagents are optionally coated. The first plate and the second plate can optionally be enclosed in a bag to facilitate shipping and extend shelf life.

  For hemocytometry, only about 1 uL (microliter) (or about 0.1 uL to 3 uL) of blood is required for a sample taken from a finger or other body location. The blood sample can be directly applied to the first plate and the second plate from the human body (eg, finger) without dilution. The blood sample is then between the inner surfaces of the first and second plates by facing the first and second plates together. If either reagent is pre-deposited (staining dye or anticoagulant), they will be deposited on the inner surface for mixing with the sample. The first plate and the second plate are then pressed by fingers or a simple mechanical device (e.g., a spring pressing clip). Under the press, the inner spacing decreases, the reduction finally stops at the value set by the height of the spacer, and reaches the final sample thickness, which is the final sample thickness. Equal to the internal spacing. Since the final internal spacing is known, the final sample thickness is known, ie quantified (measured) in this way.

  If the blood sample is not diluted, then after pressing (sample deformation), the thickness of the spacer and the final sample is thin, eg less than 1 μm, less than 2 μm, less than 3 μm, less than 4 μm. Small, less than 5 μm, less than 7 μm, less than 10 μm, less than 15 μm, less than 20 μm, less than 30 μm, less than 40 μm, less than 50 μm, less than 60 μm, less than 80 μm Smaller, smaller than 100 μm, smaller than 150 μm, or in any range between any two of these values. If the final sample is thick, many red blood cells can overlap during imaging, which can lead to inaccurate cell counts, so a thin final sample can be useful. There is a nature. For example, undiluted whole blood with a thickness of about 4 μm gives about one layer of blood red blood cells.

  After pressing, the sample may be imaged by the smartphone directly or via additional optics (eg required lenses, filters, or light sources). The image of the sample is processed to identify cell types and cell numbers. The image processing can be done locally on the same smartphone that takes the image or remotely, but the final result is sent to the smartphone (the image is sent to a remote location and processed there). ). The smartphone indicates the cell number of the specific cell. In some cases, displaying specific advice. The advice can be stored on the smartphone prior to the examination or can come from a remote machine or an expert.

  In certain embodiments, the methods and apparatus described in Section 5 (Reagent Mixing) are used to deposit the reagents on the inner surface of the first plate and/or the second plate.

  Devices or methods for blood testing include (a) the device or method in the paragraphs described herein, and (b) the plate spacing in the closed configuration (ie, the distance between the inner surfaces of two plates) or its Including the use of such spacing, the undiluted whole blood in the plate spacing has an average lateral intercellular distance of red blood cells (RBCs) greater than the average diameter of the disc-shaped RBCs.

  Devices or methods for aligning the orientation of non-spherical cells include (a) the devices or methods described herein and (b) the plate spacing in a closed configuration (ie, between the inner surfaces of two plates). Distance) or the use of such spacing, where the spacing is less than the average size of the cells in its lengthwise direction, which is the direction of greatest dimension of the cells. Such an array can improve the measurement of sample volume (eg, red blood cell volume).

  In the present invention, the analyte in the blood test contains protein markers, a list of protein markers can be found on the American Society of Clinical Chemistry website).

26 Packaging Another aspect of the present invention relates to packaging that extends the life and ease of use of the reagents used.

  In some embodiments, the plates in CROF with or without reagents are placed in a package, one plate in one package or one or more plates in one package. In one embodiment, the first plate and the second plate are packaged in different packages before use.

  In some embodiments, the different assays share a common first plate or a common second plate. In some embodiments, each package is hermetically sealed. In some embodiments, the seal is used to prevent air, chemicals, moisture, contaminants, or any combination thereof outside the package from entering the interior of the package. In some embodiments, the package is vacuum sealed or filled with nitrogen gas or internal gas. In some embodiments, materials that can extend the shelf life of the plate and/or reagents (including capture agents, detection agents, etc.) are packaged with the plate in a package.

  In some embodiments, the packaging material is in the form of a thin layer so that the package can be easily torn by a human hand.

27 Poc, smartphone, and network One aspect of the present invention relates to a method of monitoring the health of a subject, the method comprising: providing a sample provided by the subject, a CROF-based device configured to exhibit an output representative of the sample. Applied to a detector, taking the detector output as input data, processing the detector output with a device configured to analyze the input data and generate a report, and receive the report. Including. Signal-enhanced detectors offer the advantage of being quick to detect, easy to read (e.g. replacing traditional large typical readers with smartphones) and low cost.

Body fluids In certain embodiments, the samples can include different liquid or solid samples. In some examples, the sample may be a body fluid sample from the subject. In some examples, a solid or semi-solid sample may be provided. These samples may include tissues and/or cells taken from the subject. The sample may be a biological sample. Examples of biological samples are blood, serum, plasma, nasal swabs, nasopharyngeal lavage fluid, saliva, urine, gastric juice, spinal fluid, tears, stool, mucus, sweat, earwax, oil, glandular secretions, cerebrospinal fluid. , Tissue, semen, vaginal fluid, tumor tissue-derived interstitial fluid, ocular fluid, spinal fluid, pharyngeal swab, respiration, hair, fingernail, skin, biopsy, placental fluid, amniotic fluid, cord blood, lymph fluid, cavity fluid , But not limited to, sputum, pus, microbiota, meconium, breast milk, and/or other excrements. The sample may include a nasopharyngeal wash. Nasal swabs, pharyngeal swabs, stool samples, hair, fingernails, ear lobes, exhaled breaths, and other solid, semi-solid, or liquid samples are processed, for example, in an extraction buffer prior to analysis for a fixed or variable amount of time. You may. In some cases, the extraction buffer or an aliquot thereof may then be treated like any other liquid sample. Examples of tissue samples of a subject include, but are not limited to, tightened tissue, muscle tissue, nerve tissue, epithelial tissue, cartilage, cancerous samples, or bone.

  In particular embodiments, the subject may be a human or non-human animal. The subject may be a mammal, a vertebrate, such as a mouse, monkey, human, livestock, sport animal, or pet. In some embodiments, the subject can be a patient. In other embodiments, the subject may be diagnosed with the disease or the subject may not be diagnosed with the disease. In some embodiments, the subject may be a healthy subject.

Device Readings As summarized above, aspects of the method include the use of a device configured to take the output of a detector as input data and process the input data to generate a report of a signal enhanced detector. Including processing the output. Any device suitable for taking the output of the detector as input data and processing the input data to generate a report can be used. In some embodiments, the device includes an optical recording device configured to obtain the output of the photodetector as input data. In some cases, the optical recording device is a camera, such as a digital camera. The term "digital camera" is any camera that includes as its main component an imaging lens system for forming an optical image, an image sensor for converting the optical image into an electrical signal, and an imaging device with other components. , Examples of such cameras are digital still cameras, digital movie cameras and webcams (ie those directly connected to the network, and those connected to the network via a device such as a personal computer having information processing capabilities). Cameras (publicly or privately connected to network-connected devices) to enable the exchange of images, including both. In one example, the input data can include video imaging that can capture changes over time. For example, a video can be acquired to provide an assessment of the dynamic change of the sample.

  In certain embodiments, the optical recording device has a lower sensitivity than that of the high sensitivity optical recording device used in research/clinical laboratory equipment. In some cases, the optical recording device used in the method comprises more than 200 times, 500 times, 1,000 times or more than the sensitivity of the high sensitivity optical recording device used in the research/clinical laboratory apparatus. It has a sensitivity 10 times or more lower than that of 100 times or more.

  In a particular embodiment, the device acquires the output of the detector by means of an adapter that forms the interface between the device and the detector. In a particular embodiment, the interface is generic and compatible with any device suitable for performing the method of the invention. Interfaces of interest include, but are not limited to, USB, Firewire, Ethernet, etc. In certain embodiments, the device obtains the output of the detector by wireless communication, including cellular, Bluetooth, WiFi, etc.

  In certain embodiments, the device may have a video display. A video display is a component by which a display page may be displayed in a user-perceptible manner, such as a computer monitor, cathode ray tube, liquid crystal display, light emitting diode display, touch pad or touch screen display, and/or visually perceptible. Other means known in the art for producing the possible outputs may be included. In certain embodiments, the device displays information, such as a report generated from input data obtained from the detector and/or processed data, to allow the information to be entered by the subject. Equipped with a touch screen.

  In certain embodiments, the device includes a vibration function that alerts the subject, for example, as a method of reporting generated when processing the output of the detector or in preparation for obtaining the output from the detector. Prepare

  In certain embodiments, the apparatus of the present invention is configured to process input data obtained from a signal enhancement detector. The device may be configured in any suitable manner to process the data in the method of the invention. In a particular embodiment, the device comprises a memory location for storage of instructions for data storage and/or data processing and/or for database storage. The data can be stored in memory in any suitable format.

  In a particular embodiment, the device comprises a processor for processing the data. In particular embodiments, the instructions for processing the data may be stored in the processor or may be stored in a separate memory location. In some embodiments, the device can include software for implementing the process.

  In certain embodiments, a device configured to process input data acquired from a detector includes a software implemented method of performing the process. Software implementation methods include image acquisition algorithms, image processing algorithms, user interface methods that facilitate interactions between users and computing devices, and act as means for data collection, transmission and analysis, communication protocols, and data processing. It may include one or more of the algorithms. In a particular embodiment, the image processing algorithm is one of particle count, LUT (lookup table) filter, particle filter, pattern recognition, morphological decision, histogram, line profile, terrain representation, binary conversion or color matching profile. One or more of

  In certain embodiments, the device is configured to display information on a video display or a touch screen display when the display page is interpreted by software resident in the device's memory. The display pages described herein are, for example, hypertext markup language (“HTML”), dynamic hypertext markup language (“DHTML”), extensible hypertext markup language (“XHTML”), extensible. Any suitable software language, such as Markup Language ("XML"), or another software language that may be used to create computer files that can be displayed on a video or other display in a manner perceptible by a user. Can be created using. Any software-readable medium having logic, code, data, instructions may be used to implement any software or step or method. If the network includes the Internet, the display page can include any suitable type of web page.

  The display page according to the present invention may be, for example, a VBSscript routine, a JScript routine, a Javascript routine, a Java applet, an ActiveX component, an ASP. It may include built-in functionality including software programs stored in a memory device such as NET, AJAX, Flash applets, Silverlight applets, or AIR routines.

  The display page includes well-known features of graphical user interface technologies such as frames, windows, scroll bars, buttons, icons, and hyperlinks, and well-known features such as "click" or touch screen interfaces. Pointing and clicking on a graphical user interface button, icon, menu option, or hyperlink is also known as "selecting" a button, option, or hyperlink. Display pages according to the present invention may also incorporate multimedia features, multi-touch, pixel sensing, IR LED-based surfaces, vision-based interaction with or without a camera.

  The user interface may be displayed on a video display and/or display page. The user interface can display a report generated based on the analytical data for the sample, as described further below.

  The processor may be configured to process the data used in the method of the present invention in any suitable manner. The data is processed into, for example, binned data, transformed data (eg, time domain data transformed into the frequency domain by a Fourier transform), or in combination with other data. The processing can put the data into a desired format and can include changing the format of the data. The processing may include detection of signals from a sample, correction of raw data based on mathematical manipulations or corrections and/or calibrations specific to the device or reagents used to test the sample, calculation of values such as concentration values, ( Comparison with baselines, thresholds, standard curves, historical data or data from other sensors, etc., determination of whether the test is accurate, above or below normal or acceptable ranges, or abnormal conditions Highlighting of values or results that are outliers or that may cause concern, or a combination of results that can also indicate the presence of an abnormal condition, curve fitting, mathematical or other analytical inference ( The use of data as a basis for deduction (including deductive, inductive, Bayesian, or other reasoning) and other suitable processing forms. In certain embodiments, the processing can include comparing the processed data with a database stored on the device to retrieve instructions for a sequence of actions performed by the subject.

  In certain embodiments, the device is configured to process the input data by comparing the input data with a database stored in memory to retrieve instructions for a sequence of actions performed by the subject. Good. In some embodiments, the database can include stored information, including thresholds for analytes of interest. The threshold may be useful in determining the presence or concentration of one or more analytes. Thresholds may be used to detect situations where alerts are useful. The data storage unit may include records or other information useful in generating a report on the sample.

  In certain embodiments, the device may be configured to acquire data that is not the output from the signal enhancement detector. Thus, in some cases, the device may be configured to acquire data that is not representative of the sample provided by the subject but is still representative of the subject. These data include, but are not limited to, age, sex, height, weight, personal and family medical history, and the like. In certain embodiments, the apparatus is configured to process input data obtained from the output of the detector in combination with data obtained independently of the output of the detector.

  In a particular embodiment, the device is a network such as a local area network (LAN), a wide area network (WAN) such as the Internet, a personal area network, a telecommunication network such as a telephone network, a mobile phone network, a mobile network, a wireless network, It may be configured to communicate via a data providing network, or any other type of network. In particular embodiments, the device may be configured to utilize a wireless technology such as Bluetooth or RTM technology. In some embodiments, the device may be configured to utilize various communication methods such as dial-up wired connections using modems, direct links such as TI, ISDN or cable lines. In some embodiments, the wireless connection may use an exemplary wireless network such as a cellular, satellite, or pager network, GPRS, or a local data transfer system such as Ethernet or Token Ring on a LAN. In some embodiments, the device is capable of wireless communication using infrared communication components.

  In a particular embodiment, the device is configured to receive a computer file transmitted from a server over a network and stored in memory. A device may receive a tangible computer-readable medium, which may include instructions, logic, data, or code, which may be stored in permanent or temporary memory of the device, or some other device It may affect the form or initiate the action. One or more devices can communicate with computer files or links that can provide access to other computer files.

  In some embodiments, the device is a personal computer, server, laptop computer, mobile device, tablet, cell phone, mobile phone, satellite phone, smartphone (eg, iPhone, Android, Blackberry, Palm, Symbian, Windows), A personal digital assistant, a Bluetooth device, a pager, a landline phone, or other network device. Such a device may be a device capable of communicating. As used herein, the term "mobile phone" uses a telephone handset that can operate in a cellular network, a Voice over IP (VoIP) network such as Session Initiation Protocol (SIP), or an 802.11x protocol. Wireless local area network (WLAN), or any combination thereof. In certain embodiments, the device is handheld and compact so that it can fit into a consumer's wallet and/or pocket (eg, pocket size).

  In certain embodiments, the method comprises transmitting the data from the sample to a remote location where the transmitted data was analyzed. The remote location may be at a different location than where the device is located. Remote locations can include, but are not limited to, hospitals, clinics or other medical facilities, or laboratories. In some examples, the remote location may also have a computer, eg, a server, configured to communicate with (ie, receive information from, and send information to, the device over a network). Good. In some embodiments, the device can send data to the cloud computing infrastructure. The device can access the cloud computing infrastructure. In some embodiments, on-demand provisioning of computing resources (data, software) may occur over a computer network rather than from a local computer. The device may contain very little software or data (perhaps only a minimal operating system and web browser) that acts as a basic display terminal connected to the Internet. Cloud-based applications and services can support any type of software application or service because the cloud can be the primary delivery mechanism. The information provided by and/or accessed by the device may be distributed in various computational resources. Alternatively, the information may be stored in one or more fixed data storage units or databases.

  In a particular embodiment, the remote location comprises a central database stored in a data storage unit that receives and analyzes data transmitted from the device. The data storage unit may store computer readable media that may include code, logic, or instructions for the processor to perform one or more steps. In some embodiments, the received data was analyzed in comparison to the data contained in the central database and the results returned to the subject. Analysis refers to correction of raw data based on mathematical manipulations or corrections and/or calibrations specific to the equipment or reagents used to test the sample, calculation of values such as concentration values, (baseline, threshold, standard curve). , Comparison with historical data, or data from other sensors, etc., determination of whether the test is accurate, abnormalities (such as above or below the normal range or acceptable range, or indicating abnormal conditions) Highlighting of values or consequences that are or can cause concern, or combination of outcomes that can together indicate the presence of abnormal conditions, curve fitting, mathematical or other analytical inference (a priori, inductive, The use of data as a basis for (including Bayesian, or other inference) and other suitable forms of processing can be included.

  In certain embodiments, the analysis may include comparing the analyzed data with a database stored in a remote data storage unit to retrieve instructions for a course of action to be performed by the subject. .. In some embodiments, the database can include stored information, including thresholds for analytes of interest. The threshold may be useful in determining the presence or concentration of one or more analytes. Thresholds may be used to detect situations where alerts are useful. The data storage unit may include any other information regarding sample preparation or clinical testing that may be performed on the sample. The data storage unit may include records or other information that may be useful for generating a report on the analyzed data.

  In certain embodiments, the healthcare professional is at a remote location. In another embodiment, the healthcare professional accesses the data sent by the device at a remote location or at a third location different from the location of the apparatus. Medical professionals may include individuals or organizations associated with a healthcare system. The health care professional may be a health care professional. The medical professional may be a doctor. A health care professional may be an individual or institution that systematically provides preventative, curative, facilitative, or rehabilitative health care services to individuals, families, and/or communities. Examples of medical professionals are doctors (including general practitioners and specialists), dentists, physician assistants, nurses, midwives, pharmacologists/pharmacists, dietitians, therapists, psychologists, shiatsu, clinical staff, physiotherapy. Trained to provide medical professionals, blood sampling specialists, occupational therapists, optometrists, emergency medical technicians, medical assistants, medical examination technicians, medical prosthesis technicians, x-ray technicians, social workers, and various healthcare services. It may include a wide variety of human resources. The medical professional may or may not be authorized to write a prescription. Medical professionals may work in or belong to hospitals, healthcare centers and other service delivery points, or academic training, research, and management. Some medical professionals may provide care and treatment services to patients in private homes. Community health workers may work outside formal health care institutions. Health care services, health record managers and health information technicians, and other support workers may be health care professionals or may belong to health care providers.

  In some embodiments, the medical professional may be familiar with or in contact with the subject. The subject may be a medical professional patient. In some examples, a medical professional may instruct a subject to undergo a clinical examination. In one example, the medical professional may be the subject's primary care physician. The medical professional may be any type of physician for the subject, including general practitioners and specialists.

  Thus, the healthcare professional can analyze or review the data transmitted from the device and/or the results of the analysis performed at the remote location. In certain embodiments, the medical professional may send instructions or recommendations to the subject based on the data sent by the device and/or the data analyzed remotely.

Method of Monitoring Subject's Health When performing the method, the sample provided by the subject is contacted with the sample-receiving area of the signal-enhanced detector using, for example, a pipette, dropper, syringe, etc. Can be applied to the signal enhancement detector by any suitable method, including: In a particular embodiment, when the signal-enhanced detector is placed on a dipstick-type support, as described below, the sample is subjected to signal-enhanced detection by immersing the sample-receiving area of the dipstick in the sample. Can be applied to vessels.

  The subject can provide any volume of sample. Examples of volumes are about 10 mL or less, 5 mL or less, 3 mL or less, 1 microliter (μL, also referred to as “uL”) or less, 500 μL or less, 300 μL or less, 250 μL or less, 200 μL or less, 170 μL or less, 150 μL or less, 125 μL Hereinafter, it can include, but is not limited to, 100 μL or less, 75 μL or less, 50 μL or less, 25 μL or less, 20 μL or less, 15 μL or less, 10 μL or less, 5 μL or less, 3 μL or less, 1 μL or less. The sample volume may be about 1 drop of sample. The amount of sample may be the amount taken from the pricked finger or fingertip. The amount of sample may be the amount taken from a microneedle or venous draw. Any volume, including those described herein, can be applied to the signal enhancement detector.

  1 or more, 2 or more, 3 or more, 4 or more, 5 or more, 6 or more, 7 or more, 8 or more, 10 or more, 12 or more, 15 or more, or 20 or more from the test subject Different types of samples can be provided. A single type of sample or multiple types of samples can be provided from a subject at the same time or at different times. A single type of sample or multiple types of samples can be provided from a subject at the same time or at different times.

  The sample from the subject may be collected once or multiple times. The data may be taken at discrete points in time, or continuously over time. The data collected over time may be aggregated and/or processed. In some examples, the data may be aggregated and used for analysis over time to facilitate screening, diagnosis, treatment and/or disease prevention.

  In a particular example, the period from application of the sample to the signal enhanced detector to producing an output that can be received by the device is from 1 second to 30 minutes, including 1 minute to 5 minutes, eg 10 seconds to 20 minutes. Minutes may range from 30 seconds to 10 minutes. In some cases, the period from application of the sample to the signal enhancement detector to generation of output receivable by the device is 1 hour or less, 30 minutes or less, 15 minutes or less, 10 minutes or less, 5 minutes or less, 3 minutes or less. 1 minute or less, 50 seconds or less, 40 seconds or less, 30 seconds or less, 20 seconds or less, 10 seconds or less, 5 seconds or less, 2 seconds or less, 1 second or less, or even shorter. In some cases, the period from application of the sample to the signal-enhanced detector to generation of output receivable by the device is 100 ms or more, including 200 ms or more, eg, 500 ms or more, 1 second or more, 10 Seconds or longer, 30 seconds or longer, 1 minute or longer, 5 minutes or longer, or even longer.

  In a particular embodiment, the method of the invention comprises processing the output of the detector with a device for generating a report. The detector output can be processed by the device to produce a report by any suitable method, as described above.

  Embodiments of the method can further include receiving a report generated by the device. Reports include viewing reports displayed on the screen of the device, viewing emails or text messages sent to the subject, listening to voice messages generated by the device, sensing vibrations generated by the device. It may be provided and received in any convenient format, including, but not limited to.

  As mentioned above, sending data to a remote location can be accomplished in any convenient manner. Such transmission may occur via electronic signals, radio frequency signals, optical signals, cellular signals, or any other type of signal transmitted via a wired or wireless connection. The transmission of any data, the description of electronic data, the transmissions described elsewhere herein include electronic signals, radio frequency signals, optical signals, cellular signals, or otherwise transmitted via a wired or wireless connection. May be done via any type of signal. The transmitted data may include input data and/or processed data and/or generated reports. The transmitted data may include data that was not obtained from the signal-enhanced detector, that is, data that is not representative of one aspect of the sample obtained from the subject, but that is representative of other aspects of the subject as described above. it can.

  In certain embodiments, the method includes receiving the analyzed data. The analyzed data can be viewed by the subject by viewing the analyzed data displayed on the screen on the device, by viewing an email or text message sent to the subject, listening to a voice message generated by the device, It may be received using any convenient method, including, but not limited to, sensing the vibrations generated by the device.

System As summarized above, aspects of the invention include a system used to perform the method. In some embodiments, the system includes a device configured to receive the output from the signal enhancement detector as input data, process the input data to generate a report, and receive the report, wherein And the signal-enhanced detector is configured to indicate the output by acquiring a sample provided by the subject and generating an output representative of the sample.

  In some embodiments, the signal enhanced detector includes a fluid handling component, such as a microfluidic fluid handling component. The fluid handling component may be configured to direct one or more fluids to a signal enhancement detector. In some examples, the fluid handling components are configured to guide fluids such as, but not limited to, sample solutions, buffers, etc. Fluid handling components include, but are not limited to, passive pumps and microfluidic channels. In some cases, passive pumps are configured for capillary action driven microfluidic handling and fluid routing through the signal enhancement detectors disclosed herein. In certain examples, the microfluidic fluid handling component delivers a small amount of fluid, such as 100 μL or less, 50 μL or less, or 25 μL or less, or 10 μL or less, or 5 μL or less, including 500 μL or less, 1 mL or less, including 1 μL or less. Is configured as follows. Therefore, in certain embodiments, no external power supply is required to operate the system.

  In certain embodiments, the signal enhancement detector has dimensions within the range of 5 mm x 5 mm to 100 mm x 100 mm, including dimensions of 50 mm x 50 mm or less, such as 25 mm x 25 mm or less, or 10 mm x 10 mm or less. In certain embodiments, the signal enhancement detector has a thickness in the range of 2 mm to 0.3 mm, or 5 mm to 0.1 mm, including 1 mm to 0.4 mm, such as 3 mm to 0.2 mm.

  In some embodiments, the signal enhancement detector can have an identifier. The identifier may be a physical object formed on the signal enhancement detector. For example, the identifier may be read by a device of the system of the invention. Thus, in some examples, the output from the signal enhancement detector may include the identifier. In some embodiments, the camera can capture an image of the identifier and the image can be analyzed to identify the signal enhancement detector. In one example, the identifier may be a barcode. The barcode may be a 1D or 2D barcode. In some embodiments, the identifier can emit one or more signals that can identify the signal enhancement detector. For example, the identifier can provide infrared, ultrasound, light, voice, electrical, or other signals that can indicate the identity of the signal enhancement detector. The identifier may utilize a radio frequency identification (RFID) tag. The identifier may be stored in the memory of the signal enhancement detector. In one example, the identifier may be a computer-readable medium.

  The identifier is a device configured to take the output from the signal enhancement detector and process the output to determine the particular type of signal enhancement detector used to generate the output representative of the sample. It can include information that enables it. In certain embodiments, the identifier provides the database with a key that associates each identifier key with information specific to the type of signal-enhanced detector used to generate the output representative of the sample. Information specific to the type of signal-enhanced detector includes identification of the analyte that the signal-enhanced detector is configured to bind to, coordinates of the location at which the particular analyte can be bound on the signal-enhanced detector, and Includes, but is not limited to, detection sensitivity for analytes and the like. The database may include other information associated with a particular signal enhancement detector, including expiration date, lot number, etc. The database may reside on a device, be provided on a computer-readable medium, or be accessible by the device on a remote server.

  In certain embodiments, the system does not have a physical signal amplification process, but is 10x or more, including 100x or more, such as 200x or more, 500x or more, 1000x or more, or higher sensitive laboratory grade reader. Has a higher detection sensitivity than the system using In certain embodiments, the system does not have a physical signal amplification process, but is 10 to 10,000 times, including 100 to 5000 times, including 200 to 2000 times, or 500 to 1000 times, a high sensitivity laboratory grade reader. Has a higher detection sensitivity than the system using

  Embodiments of the system include a device configured to generate a report and provide the report to a subject in processing the output from the signal enhancement detector. In some embodiments, the report can include diagnostic information about the subject for a condition such as a disease. In certain embodiments, the system achieves a diagnostic accuracy of 85% or more, or 75% or more, including 90% or more, for example 80% or more.

Utility The methods and systems of the present invention find use in a variety of different applications where it is desired to determine the presence and/or quantification of one or more analytes in a sample, and/or to monitor the health of an individual. For example, the systems and methods of the invention are used to detect proteins, peptides, nucleic acids and the like. In some cases, the systems and methods of the invention are used to detect proteins.

  In some embodiments, the systems and methods of the present invention are used to detect nucleic acids, proteins, or other biomolecules in a sample. The method can include the detection of a set of biomarkers in the sample, eg, two or more distinct protein biomarkers. For example, the method may be used for rapid clinical detection of two or more disease biomarkers in a biological sample, such as for diagnosing a disease state in a subject, or for ongoing management or treatment of a disease state in a subject. . As noted above, communication with a physician or other healthcare provider will ensure that the physician or other healthcare provider is aware and aware of potential concerns, and thus may take appropriate action. Sex can be higher.

  In certain embodiments, the systems and methods of the invention are used for biomarker detection. In some cases, the systems and methods of the invention include the presence or absence of particular biomarkers and blood, plasma, serum, or, without limitation, urine, blood, serum, plasma, saliva, semen, prostatic fluid, teat aspirate. Increase or decrease the concentration of certain biomarkers in other body fluids or excretions such as, tears, sweat, feces, buccal swabs, cerebrospinal fluid, cell lysate samples, amniotic fluid, gastrointestinal fluid, biopsy tissue, etc. It may be used for detecting.

  The presence or absence of a biomarker or a significant change in biomarker concentration may be used to diagnose disease risk, the presence of disease in an individual, or the adjustment of treatment of the disease in an individual. For example, the presence of a particular biomarker or biomarker group influences the choice of drug treatment or regimen given to an individual. When assessing potential drug therapies, biomarkers can be used as alternatives to natural endpoints such as survival or irreversible disease. If the treatment modifies a biomarker that is directly related to improved health, then the biomarker can be used as an alternative endpoint to assess the clinical benefit of a particular treatment or dosing regimen. Thus, personalized diagnosis and treatment based on a particular biomarker or panel of biomarkers detected in an individual is facilitated by the systems and methods of the invention. Moreover, as mentioned above, early detection of disease-related biomarkers is facilitated by the high sensitivity of the devices and systems of the present invention. Due to the ability to detect multiple biomarkers on mobile devices such as smartphones combined with sensitivity, scalability and ease of use, the systems and methods of the present disclosure can be used for portable and point-of-care or bedside molecular diagnostics. Be done.

  In certain embodiments, the systems and methods of the invention are used to detect biomarkers for diseases or disease states. In some cases, the systems and methods of the invention are used to characterize cell signaling pathways and detect biomarkers for intracellular communication for drug discovery and vaccine development. For example, the systems and methods of the present invention may be used to detect and/or quantify the amount of biomarkers in pathological, healthy, or benign samples. In certain embodiments, the systems and methods of the invention are used to detect biomarkers for infectious diseases or disease states. In some cases, the biomarker may be a molecular biomarker, including but not limited to proteins, nucleic acids, carbohydrates, small molecules and the like.

  The systems and methods of the present invention may be performed using the detection and/or quantification of biomarkers as described above, screening assays that test samples at regular intervals for asymptomatic subjects, the presence and/or amount of biomarkers. It is used in diagnostic assays such as prognostic assays that predict the course of certain diseases, stratified assays that can predict a subject's response to different drug treatments, and efficacy assays in which the effectiveness of drug treatment is monitored. Not limited.

  The system and method of the present invention may also be used in validation assays. For example, validation assays can be used to validate or confirm that a potential disease biomarker is a reliable indicator of the presence or absence of disease across many individuals. The assay times of the systems and methods of the present invention are short, which can facilitate increased throughput for screening multiple samples in the shortest amount of time.

  In some examples, the system and method of the present method can be used without the need for a laboratory setting for practice. Compared to comparable analytical laboratory equipment, the apparatus and system of the present invention provide comparable analytical sensitivity in portable handheld systems. In some cases, weight and operating costs are lower than in typical fixed laboratory equipment. In addition, the systems and methods of the present invention can be used in a home environment for home testing without physician prescription by untrained persons to detect one or more analytes in a sample. .. The system and apparatus of the present invention may also be used for rapid diagnosis in clinical environments, such as bedside, or environments where fixed lab equipment is not installed due to cost or other reasons. Good.

Kits Aspects of the invention include a kit that provides a signal-enhanced detector for monitoring the health of a subject, and instructions for performing the method of the invention using a handheld device, such as a cell phone. These instructions may be present in the kit in various forms, one or more of which may be present in the kit. One form of these instructions for use is as information printed on a suitable medium or substrate, such as one or more sheets of printed information, present on kit packaging, package inserts, etc. Good. Another means is a computer-readable medium on which information is recorded or stored, such as a floppy disk, CD, DVD, Blu-ray, computer-readable memory or the like. Yet another means that may exist is the address of the website where the information at the deleted site may be accessed via the Internet. The kit can further include software provided on a computer-readable medium for performing the method for monitoring the health of a subject on a device, as described herein. Any convenient means may be present in the kit.

Samples, Health, and Uses Samples from subjects, health of subjects, and other uses of the invention are described further below. Exemplary samples, health conditions, and uses are also described, for example, in U.S. Patent Application Publications 2014/0154668 and 2014/0045209, which are hereby incorporated by reference.

  The present invention finds use in a variety of applications, such applications generally involving analytes in which the presence of a particular analyte in a given sample is at least qualitatively, if not quantitatively detected. It is a detection application. Protocols for performing analyte detection assays are well known to those of skill in the art and need not be discussed at length here. Generally, the sample suspected of containing the analyte of interest is contacted with the surface of the nanosensor of the invention under conditions sufficient for the analyte to bind to each capture agent attached to the sensor. .. The capture agent has a very specific affinity for the target molecule of interest. This affinity may be an antigen-antibody reaction in which the antibody binds to a specific epitope on the antigen, or a DNA/RNA or DNA/RNA hybridization reaction that is sequence-specific between two or more complementary strands of nucleic acid. Good. Thus, if the analyte of interest is present in the sample, it is likely that it will bind to the sensor at the site of the capture agent and a complex will be formed on the surface of the sensor. That is, the captured analyte is immobilized on the surface of the sensor. Then, after removal of unbound analyte, the presence of this bound complex on the surface of the sensor (ie, the immobilized analyte of interest) is removed, eg, using a labeled secondary capture agent. To be detected.

  Specific analyte detection applications of interest include hybridization assays in which nucleic acid capture agents are used, and protein binding assays in which polypeptides, such as antibodies, are used. In these assays, a sample is first prepared and, after sample preparation, the sample is contacted with a nanosensor of the invention under specific binding conditions, whereby the complex is a target complementary to the capture agent attached to the surface of the sensor. It is formed between nucleic acids or polypeptides (or other molecules).

  In one embodiment, the capture oligonucleotide is synthetic single-stranded DNA thiolated at one end that is 20-100 bases in length. These molecules are immobilized on the surface of the nanodevice and capture target single-stranded DNA (which may be at least 50 bp long) having a sequence complementary to the immobilized capture DNA. After the hybridization reaction, detection single-stranded DNA whose sequence is complementary to the unoccupied nucleic acid of the target DNA (which may be 20-100 bp in length) is added to hybridize with the target. The detection DNA has one end bound to a fluorescent label and its emission wavelength is within the plasmon resonance of the nanodevice. Therefore, by detecting the fluorescence emission emitted from the surface of the nanodevice, the target single-stranded DNA can be accurately detected and quantified. The length of the captured and detected DNA determines the melting temperature (nucleotide strands separate above the melting temperature), the extent of mispairing (the longer the strand, the less mispairing). One of the concerns of choosing complementary bond lengths depends on the need to minimize mismatches while keeping the melting temperature as high as possible. Moreover, the total length of the hybridization length is determined to achieve optimal signal amplification.

  The sensor of the present invention comprises the steps of (a) obtaining a liquid sample from a patient suspected of having a disease or condition, and (b) contacting the sample with the nanosensor of the present invention, wherein the capture agent of the nanosensor is a biomolecule of a disease. A step of specifically binding the marker and contacting under conditions suitable for the specific binding of the biomarker and the capture agent, (c) removing any biomarker not bound to the capture agent , And (d) a method of diagnosing a disease or condition comprising the step of reading an optical signal from a biomarker that remains bound to the nanosensor, wherein the optical signal is used by the patient to diagnose the disease or condition. The method further comprises labeling the biomarker with a luminescent label, either before or after binding to the capture agent. As described in more detail below, the patient is suspected of having cancer and the antibody binds to a cancer biomarker. In other embodiments, the patient is suspected of having a neurological disorder and the antibody binds to a biomarker of neurological disorder.

  Application of the sensor of the present invention includes (a) infectious diseases and parasitic diseases, injuries, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, and organic diseases such as lung diseases and renal diseases. Detection, purification, and quantification of compounds or biomolecules that correlate with stages; Quantification, (c) detection of chemical substances or biological samples that pose a risk to food safety or national security such as hazardous waste, anthrax, etc., quantification, (d) glucose, blood oxygen level, total blood cell count, etc. Quantification of biological parameters in medical or physiological monitors, (e) detection and quantification of specific DNA or RNA from biological samples such as cells, viruses, body fluids, (f) in chromosomes and mitochondria for genomic analysis This includes, but is not limited to, sequencing and comparison of gene sequences in DNA, or (g) detection of reaction products such as during synthesis or purification of pharmaceuticals.

  Detection can be carried out in various sample matrices such as cells, tissues, body fluids, and feces. The target body fluids are amniotic fluid, aqueous humor, vitreous humor, blood (eg, whole blood, fractionated blood, plasma, serum, etc.), breast milk, cerebrospinal fluid (CSF), earwax, chyle, chyme, Endolymph, perilymph, feces, stomach acid, gastric juice, lymph, mucus (including nasal discharge and mucus), pericardial fluid, peritoneal fluid, pleural fluid, purulent fluid, mucous secretions, saliva, sebum (skin oil), semen , Sputum, sweat, synovial fluid, tears, vomiting, urine, and exhaled breath condensate.

  In some embodiments, the biosensor of the present invention can be used to diagnose a pathogen infection by detecting a target nucleic acid from a pathogen in a sample. Target nucleic acids include, for example, human immunodeficiency virus 1 and 2 (HIV-1 and HIV-2), human T-cell leukemia virus and 2 (HTLV-1 and HTLV-2), respiratory syncytial virus (RSV), adenovirus. Virus, hepatitis B virus (HBV), hepatitis C virus (HCV), Epstein-Barr virus (EBV), human papillomavirus (HPV), varicella zoster virus (VZV), cytomegalovirus (CMV), herpes simplex virus. 1 and 2 (HSV-1 and HSV-2), human herpesvirus 8 (HHV-8, also known as Kaposi's sarcoma herpesvirus), and yellow fever virus, Japanese encephalitis virus, West Nile virus, and Ebola virus It may be from a virus selected from the group consisting of flaviviruses. However, the invention is not limited to the detection of nucleic acids, such as DNA or RNA, sequences of the above viruses, and can be applied without problems to other important pathogens in veterinary and/or human medicine.

  Due to DNA sequence homology, human papillomavirus (HPV) is further subdivided into over 70 different types. These types cause various diseases. HPV types 1, 2, 3, 4, 7, 10, and 26-29 cause benign warts. 5, 8, 9, 12, 14, 15, 17, 19-25, and 46-50 HPV cause lesions in patients with compromised immune systems. Types 6, 11, 34, 39, 41-44 and 51-55 cause benign pointed warts on the genital area and airway mucosa. HPV types 16 and 18 cause special epithelial dysplasia of the reproductive mucosa and are associated with a high proportion of invasive cancers of the cervix, vagina, vulva, and anal canal, making it of special medical interest. It is a thing. Human papillomavirus DNA integration is believed to be crucial in carcinogenesis of cervical cancer. Human papillomavirus can be detected, for example, from the DNA sequences of its capsid proteins L1 and L2. Therefore, the method of the present invention is particularly applicable to detecting 16 and/or 18 HPV DNA sequences in tissue samples in order to assess the risk of developing cancer.

  In some cases, nanosensors can be used to detect biomarkers that are present in low concentrations. For example, nanosensors detect cancer antigens in easily obtained body fluids (eg, blood, saliva, urine, tears, etc.), and biosensors of easily obtained body fluids (eg, neuropathy (eg, Alzheimer's antigen)). Marker) for the detection of tissue-specific disease biomarkers, for detection of infections (especially for detection of low titer latent viruses such as HIV), for detection of fetal antigens in maternal blood, and for example in subjects. It may be used for the detection of exogenous compounds (eg drugs or contaminants) in the bloodstream.

  The table below provides a list of protein biomarkers that may be detected using the nanosensors of the present invention (when used with appropriate monoclonal antibodies) and their associated diseases. One of the potential sources of biomarkers (eg "CSF", cerebrospinal fluid) is also shown in the table. In many cases, the biosensor of the present invention can detect biomarkers in different displayed body fluids. For example, biomarkers found in cerebrospinal fluid (CSF) can be identified in urine, blood, or saliva.

  Health conditions diagnosed or measured by the present method, apparatus, and system include chemical balance, nutritional health, exercise, fatigue, sleep, stress, prediabetes, allergy, aging, environmentally harmful substances, pesticides, and herbicides. , Exposure to synthetic hormone analogs, pregnancy, menopause, and male quiescence.

  In certain embodiments, relative levels of nucleic acids in two or more different nucleic acid samples may be obtained and compared using the methods described above. In these embodiments, the results obtained from the above methods are usually normalized and compared to the total amount of nucleic acid (eg, constitutive RNA) in the sample. This may be done by comparison to a ratio or other means. In certain embodiments, the nucleic acid profiles of two or more different samples can be compared to identify nucleic acids associated with a particular disease or condition.

  In some examples, the different samples may be composed of an "experimental" sample, ie, a sample of interest and a "control" sample that is comparable to the experimental sample. In many embodiments, the different samples are pairs of cell types or fragments thereof, one cell type being the cell type of interest, such as an abnormal cell and the other cell type being a control cell type, For example, normal cells. When comparing two fragments of a cell, the fragment is usually the same fragment from each of the two cells. However, in certain embodiments, two fragments of the same cell can be compared. Exemplary cell type pairs are, for example, tissue biopsies (eg, colon, breast, prostate, lung, tissue with a disease such as skin cancer, or tissue infected with a pathogen), usually from the same patient. Cells isolated from and from normal cells from the same tissue and infected or treated by pathogens (eg, peptides, hormones, altered temperature, growth conditions, physical stress, cell transformation, etc. Environmental or chemical factors) Cells grown in immortal tissue culture (eg cells with a proliferative mutation or immortalizing transgene) and normal cells (eg not immortal, uninfected or treated). Cells that are identical to the experimental cells, except for the absence of cancer, disease, an aged mammal, or a mammal exposed to a condition, and a mammal of the same species, preferably healthy or young. Includes cells isolated from a mammal having cells of mammalian origin, a differentiated cell and an undifferentiated cell from the same mammal (eg, a precursor cell of another cell in the mammal) .. In one embodiment, different types of cells may be used, eg, neuronal and non-neuronal cells, or cells in different states (eg, before and after stimulation on the cells). In another embodiment of the invention, the experimental material is a cell susceptible to infection by a pathogen, such as a virus such as human immunodeficiency virus (HIV), and the reference material is resistant to infection by the pathogen. Cells that have. In another embodiment of the invention, the sample pair is represented by undifferentiated cells and differentiated cells such as stem cells.

Solid-Phase Assay In a surface-immobilized assay that captures the target analyte in the sample with a capture agent immobilized on the surface of the first plate (ie, the solid phase), a saturated incubation to capture the target analyte, typically from the sample. Short times and/or short saturation incubation times for immobilizing capture agents, detection agents or other entity partners in solution on the first plate surface are desirable.

  Furthermore, in surface-immobilized and other assays, other reagents are rapidly added to the sample and mixed to bind to the target entity in the sample, i.e. to reduce the time required for good mixing. There is a need to.

  One aspect of the present invention relates to devices, systems, and methods that can reduce saturation incubation times by reducing and/or controlling the thickness of the sample or liquid and/or by making the sample thickness uniform. .. One important advantage of the present invention is that it is fast, simple and low cost. For example, the present invention simplifies that by allowing the saturation incubation time to be greater than or equal to that of the microfluidic channel-based assay without the use of any complex prefabricated microfluidic channels and pumps, Easy to use and low cost.

  One basic principle of the invention is that in surface immobilization assays, the saturation incubation time for immobilization of the target analyte/entity at the binding site when the size of the binding site area (minimum) is larger than the sample thickness. Is based primarily on the fact that the target analyte/entity in the sample is defined primarily by the time it takes for the sample thickness distance to diffuse (ie the analyte/entity diffusion time). The diffusion time of an entity over a distance in a sample is proportional to the square of the distance and inversely proportional to the diffusion constant of the entity. Therefore, by reducing the thickness of the sample, the diffusion time is significantly reduced.

28 Controlling and Measuring Sample Thickness without Spacers In some embodiments of the invention, the spacers used to adjust the sample or sample of related volume include (a) spacing between plates. And a device capable of controlling the plate position and moving the plates to the desired interplate spacing based on the information provided by the sensors. In some embodiments, all spacers are replaced by translation stages, monitoring sensors and feedback systems.

  Measuring Distance and/or Sample Thickness Using Optical Methods In some embodiments, measuring the distance between inner surfaces (f) comprises using optical interference. Optical interference can use multiple wavelengths. For example, an optical signal due to interference of light reflected on the inner surfaces of the first plate and the second plate vibrates at the wavelength of light. From the vibration, the spacing between the inner surfaces can be determined. One or both inner surfaces can be coated with a light-reflecting material to enhance the interfering signal.

  In some embodiments, the measurement (f) of the spacing between the inner surfaces involves capturing an optical image (eg, a 2D (two-dimensional)/3D (three-dimensional) image of the sample, the imaging at different viewing angles, Different wavelengths, different phases, and/or different polarizations) and image processing.

  Measuring the Area or Volume of the Entire Sample Using Optical Methods In some embodiments, measuring the area or volume of the entire sample (f) involves optical imaging (eg, 2D (2D)/3D(3) of the sample). (Dimensional) image is captured, which can be performed at different viewing angles, different wavelengths, different phases, and/or different polarizations) and image processing. The area of the sample refers to the area in a direction substantially parallel to the first plate and the second plate. 3D imaging can use the method of fringe projection profilometry (FPP), which is one of the most popular methods for acquiring a three-dimensional (3D) image of an object.

  In some embodiments, measuring the area or volume of a sample by imaging includes (a) calibrating an image scale using a sample of known area or volume (e.g., the imager is a smartphone, The dimensions of the image taken with the smartphone can be calibrated by comparing the image taken with the same smartphone to an image of a sample of known dimensions), (b) the image and the first and second plates or Comparing scale markers (rulers) placed in its vicinity (discussed further herein), and (c) including combinations thereof.

  As used herein, light can include visible light, ultraviolet light, infrared light, and/or near infrared light. The light can include wavelengths within the range of 20 nm to 20,000 nm.

29 Other Description of Embodiment
The following methods, devices, and systems are provided. These embodiments can be implemented using any of the components, materials, parameters or steps described above or below. The following embodiment uses a CROF plate.
Embodiment 1.
(A) obtaining a sample containing the analyte,
(B) obtaining a first plate and a second plate that are movable relative to each other in different configurations, each plate having a substantially flat sample contact surface; One or two plates are flexible and one or two of said plates contain spacers, which are fixed to their respective sample contact surfaces, said spacers having a predetermined substantially uniform height. And having a predetermined constant distance between spacers that is at least about twice as large as the analyte size and up to 200 μm (micrometers),
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the open configuration is such that the two plates are partially or completely separated and A configuration in which the distance between the plates is not adjusted by the spacer, a step,
(D) after (c) compressing at least a portion of the sample with the two plates into a layer of substantially uniform thickness, the uniform thickness of the plates Limited by the sample contact surface of the layer, the uniform thickness of the layer is adjusted by the spacer and the plate, and the compression is
Uniting the two plates, and
Appropriately pressing one area of at least one of said plates in parallel or sequentially to press said plates together into a closed configuration, said appropriate pressing being applied to said at least a portion of said sample. A substantially uniform pressure on the plate is generated such that the pressing causes the at least a portion of the sample to spread laterally between the sample contacting surfaces of the plate and the closed configuration to provide a uniform thickness region. The distance between the plates in the layer is adjusted by the spacer.
Including steps, and
(E) analyzing the analyte in the layer of uniform thickness while the plate is in the closed configuration.
A method for analyzing a liquid sample, comprising:
A suitable press is a method of making the pressure applied to a certain region substantially constant regardless of the shape change of the outer surface of the plate,
The method wherein the parallel press simultaneously applies pressure to a desired region and the sequential press applies pressure to a portion of the desired region and gradually transitions to another region.
Embodiment 2.
First plate and second plate
Including,
i. The plates are movable with respect to each other in different configurations,
ii. One or two of the plates is flexible,
iii. Each plate has a sample contact area on its respective surface for contacting a sample containing analyte,
iv. One or two of the plates include spacers secured to respective sample contact areas, the spacers having a predetermined substantially uniform height and at least about two times greater than the size of the analyte. Having a predetermined constant distance between spacers of up to 200 μm, wherein at least one spacer is within the sample contact area,
One configuration is an open configuration in which the two plates are separated and the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates, and
Another configuration is a closed configuration, which was configured after the sample was deposited in an open configuration, where at least a portion of the sample was compressed by two plates into a layer of very uniform thickness, An apparatus for analyzing a liquid sample, wherein the uniform thickness of said layer is limited by the sample contacting surface of the plate and regulated by spacers and plates.
Embodiment 3.
(A) obtaining a blood sample,
(B) obtaining a first plate and a second plate movable relative to each other in a different configuration, each plate having a substantially flat sample contact surface Or the two plates are flexible and one or two plates include spacers fixed to their respective sample contact surfaces, the spacers comprising:
i. A predetermined substantially uniform height,
ii. A pillar shape having a substantially uniform cross section and a flat upper surface;
iii. A width to height ratio of 1 or greater,
iv. A predetermined constant distance between the spacers in the range of 10]m to 200]m;
v. Filling rate of 1% or more,
vi. The product of the spacer filling rate and Young's modulus of 2 MPa or more
Having a step,
(C) depositing a blood sample on one or two plates when the plates are configured in an open configuration, the open configuration comprising two plates partially or completely separated and between the plates. The spacing is not adjusted by the spacer, the steps are:
(D) after (c), using two plates, compressing at least a portion of the blood sample into a layer of substantially uniform thickness, wherein the substantially uniform thickness is Limited by the sample contact surface of the plate, the uniform thickness of the layer is adjusted by the spacer and the plate and has an average value in the range 1.8]m-3]m with a variation of less than 10%. And wherein the compression is
Uniting the two plates, and
In order to press the plates together into a closed configuration, one or more regions of at least one of the plates are suitably pressed in parallel or sequentially and by said appropriate pressing through said at least part of said sample. A substantially uniform pressure is generated on the plate, the press causes the at least a portion of the sample to spread laterally between the sample contacting surfaces of the plate, and the closed configuration defines a uniform thickness region of the sample. The distance between the plates in a layer is adjusted by the spacer
Including steps, and
(E) analyzing blood in a layer of uniform thickness while the plate is in the closed configuration
A method for analyzing a blood sample, comprising:
The fill factor is the ratio of the spacer contact area to the total plate area,
The suitable pressing is a method for making the pressure applied to a certain region substantially constant regardless of the shape change of the outer surface of the plate, and
The parallel press applies pressure to a desired area at the same time, and the sequential press applies pressure to a part of a predetermined area, and gradually shifts to another area.
Embodiment 4.
First plate and second plate
Including,
v. The plates are movable with respect to each other in different configurations,
vi. One or two plates are flexible,
vii. Each plate has on its respective surface a sample contact area for contacting a blood sample,
viii. One or two plates include spacers fixed to the respective plates, the spacers having a predetermined substantially uniform height and a predetermined constant spacing within a range of 7 m to 200 m. With a distance of at least one spacer in the sample contact area,
One configuration is an open configuration in which the two plates are separated and the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates, and
Another configuration is a closed configuration, which was configured after the sample was deposited in an open configuration, where at least a portion of the sample was compressed by two plates into a layer of very uniform thickness, The uniform thickness of the layer is limited by the inner surfaces of the two plates, regulated by spacers and plates, and has a mean value in the range of 1.8]m to 3]m with small variations. A device for analyzing a sample.
Embodiment 5. A method for locally binding a target entity in a portion of a liquid sample, the method comprising:
(A) obtaining a sample containing a target entity capable of diffusing into the sample,
(B) a step of obtaining a first plate and a second plate that are movable relative to each other in different configurations, one or two plates being fixed on each plate The first plate includes a spacer having a substantially uniform height, the first plate includes a binding site on its surface, the binding site has a predetermined region, and binds to the target entity to immobilize the target entity. , Step,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the open configuration is such that the two plates are partially or completely separated and The distance between the plates is not adjusted by the spacer, a configuration step,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, wherein the closed configuration compresses at least a portion of the sample into a layer of uniform thickness, The layer contacts the inner surfaces of the two plates, is bounded by the inner surfaces and contacts the binding site, the uniform thickness of the layers being regulated by the spacer and the plate is less than 250 μm and the binding A step that is substantially smaller than the linear dimension of a predetermined area of the site,
(E) After (d) and while the plate is in the closed configuration,
(1) incubating the sample for a relevant length of time and then stopping the incubation, or
(2) Incubating the sample for a time greater than or equal to the minimum relevant time length and then assessing the binding of the target entity to the binding site within a time less than or equal to the maximum relevant time length.
And
The relevant length of time is
i. More than the time required for the target entity to diffuse through the thickness of the layer of uniform thickness in the closed configuration, and
ii. Significantly shorter than the time required for the target entity to spread laterally across the smallest lateral dimension of the binding site,
At the end of the incubation in (1) or during the evaluation in (2), most of the target entity bound to the binding site comes from a relevant volume of sample,
The incubation allows the target entity to bind to the binding site and the volume of interest is the portion of the sample at the binding site in the closed configuration, step
Including the method.
Embodiment 6. A device for locally binding a target entity to a portion of a liquid sample, comprising:
First plate and second plate
Including,
i. The plates are movable relative to each other in different configurations, one or two plates being flexible,
iii. Each plate has on its respective surface a sample contact area for contacting a sample containing entities capable of diffusing into the sample,
iv. One of the plates has a binding site immobilized on its sample contact region, the binding site has a predetermined region, and the binding site bound to the target entity and immobilizing it. Have,
v. One or two plates include spacers secured to each plate, the spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers, wherein at least one spacer is , In the sample contact area,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates, and
Another configuration is a closed configuration, which was configured after the sample was deposited in an open configuration, in which at least a portion of the sample was compressed by the two plates into a layer of uniform thickness to provide a uniform thickness. At least a portion of the layer of thickness is above the binding site, and the uniform thickness of the layer is limited by the inner surfaces of the two plates, regulated by the spacer and the plate, less than 250 μm, and the binding site A device substantially smaller than the average linear dimension of a given area of the device.
Embodiment 7. A method of locally releasing a reagent into a portion of a liquid sample
(A) obtaining a sample,
(B) obtaining a first plate and a second plate moveable with respect to each other so as to have different configurations,
(I) one or two plates include spacers fixed to each plate,
(Ii) the spacer has a predetermined uniform height,
(Iii) The first plate includes a storage site on the surface thereof, the storage site having a predetermined region and containing a reagent, and the reagent is dissolved in the sample upon contact with the sample, Spread, step,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the open configuration is such that the two plates are partially or completely separated and The distance between the plates is not adjusted by the spacer, a configuration step,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, wherein the closed configuration compresses at least a portion of the sample into a layer of uniform thickness, A structure in which the layer is confined by the inner surfaces of the two plates and covers the storage site, the uniform thickness of the layer being controlled by the spacer and the plate being less than 250 μm and of a predetermined area of the storage site. Step, which is substantially smaller than the linear dimension
(E) incubating the sample for a relevant length of time after step (d) and while the plate is in the closed configuration, and then stopping the incubation,
The relevant length of time is
i. More than the time required for the target entity to spread over the thickness of the layer of uniform thickness in the closed configuration, and
ii. Less than the time required for the target entity to diffuse laterally over the linear dimension of a given region of the binding site,
Thereby, after incubation, most of the reagents initially at the storage site are in the relevant volume of the sample,
Incubation is the process of allowing the reagents to bind or mix with the sample, the volume of interest being the portion of the sample at the binding site in the closed configuration.
Including the method.
Embodiment 8. A device for locally releasing a reagent to a portion of a liquid sample, comprising:
First plate and second plate
Including,
i. The plates are movable with respect to each other in different configurations,
ii. One or two of the plates is flexible,
vi. Each plate has a sample contact area on its respective surface for contacting the sample,
vii. One of the plates includes a storage site in its sample contact area, said storage site having a predetermined area and containing a reagent, which upon contact with the sample dissolves in the sample and diffuses into the sample. And binds to the target entity,
viii. One or two of the plates include a spacer fixed to each plate, the spacer being (a) 250 m or less and being substantially smaller than the average linear dimension of a predetermined region of the reagent site. And (b) a predetermined constant distance between the spacers in the range of 200 m or less, wherein at least one spacer is in the sample contact area,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates,
Another configuration is a closed configuration, which was configured after the sample was deposited in an open configuration, in which at least a portion of the sample was compressed by the two plates into a layer of uniform thickness to provide a uniform thickness. An apparatus in which at least a portion of the layer of thickness is above the binding site, and the uniform thickness of the layer is limited by the inner surfaces of the two plates and regulated by spacers and plates.
Embodiment 9. A method for reducing the time to bind a target entity in a relevant volume of a sample to a binding site on a plate surface, the method comprising:
(A) obtaining a sample containing a target entity capable of diffusing into the sample,
(B) Obtaining a first plate and a second plate movable relative to each other in different configurations, wherein one or two plates are spacers fixed on the respective plates. Including one or two plates that are flexible, the spacers have a predetermined substantially uniform height, and a predetermined constant distance between the spacers, and the first plate is bonded to its surface. A binding site having a predetermined region and binding to and immobilizing a target entity,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the open configuration is such that the two plates are partially or completely separated and The distance between the plates is not adjusted by the spacer, a configuration step,
(D) After (c), compressing the sample by placing the two plates in a closed configuration, wherein the closed configuration is such that the thickness of the sample of the relevant volume is the same as when the plate was in the open configuration. A layer of substantially uniform thickness which is reduced in comparison, wherein the substantially uniform thickness is at least 1 mm ^Two A lateral surface area limited by the inner surfaces of the two plates and covering the binding site, the uniform thickness of said layer being adjusted by the spacer and the plate to be less than 250 μm and of the binding site A volume that is substantially smaller than the linear dimension of a given region and is of interest is part or all of the volume of the sample.
Including,
The method, wherein reducing the thickness of the sample in the relevant volume reduces the time for the binding between the binding site and the target entity in the relevant volume to reach equilibrium.
Embodiment 10. A device for locally binding a target entity to a portion of a liquid sample, comprising:
First plate and second plate
Including,
i. The plates are movable relative to each other in different configurations, one or two plates being flexible,
iii. Each plate has a sample contact area on its respective surface for contacting the sample, the sample contact area comprising an entity capable of diffusing into the sample,
iv. One of the plates has a binding site on its sample contact area, the binding site has a predetermined area and binds to and immobilizes the target entity,
v. One or two plates include spacers secured to each plate, the spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers, wherein at least one spacer is , In the sample contact area,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates, and
Another configuration is a closed configuration, which was configured after the sample was deposited in an open configuration, in which at least a portion of the sample was compressed by the two plates into a layer of uniform thickness to provide a uniform thickness. At least a portion of the layer of thickness is above the binding site, and the uniform thickness of the layer is limited by the inner surfaces of the two plates and regulated by the spacers and plates, less than 250 μm, of the binding site. Substantially smaller than the average linear dimension of a given area,
Decreasing the thickness of the sample in the relevant volume reduces the time for the binding between the binding site and the target entity in the relevant volume to reach equilibrium.
Embodiment 11. A method for parallel multiplexed assay of a liquid sample without fluidic isolation, comprising:
(A) obtaining a sample containing one or more target analytes that can diffuse into the sample,
(B) obtaining a first plate and a second plate moveable with respect to each other so as to have different configurations,
i. One or two plates including spacers fixed to the respective plates, one or two plates being flexible,
ii. The spacers have a predetermined substantially uniform height and a predetermined constant distance between the spacers,
iii. The first plate has on its surface one or more binding sites each having a predetermined region containing a capture agent that binds and immobilizes the corresponding target analyte of (a), and
iv. The second plate includes on its surface one or more corresponding storage sites, each storage site having a predetermined area, and a concentration that dissolves in the sample upon contact with the sample and diffuses into the sample. Including the detection agent of
Each capture agent, target analyte, and corresponding detection agent can form a capture agent-target analyte-detection agent sandwich at the binding site of the first plate,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the open configuration is such that the two plates are partially or completely separated and The distance between the plates is not adjusted by the spacer, a configuration step,
(D) after (c), compressing the sample by placing the two plates in a closed configuration, the closed configuration comprising:
i. At least a portion of the sample is compressed into a layer of uniform thickness, said layer contacting and constrained by the inner surfaces of the two plates, and having one or more binding sites and one or more Contact the storage site of
ii. One or more corresponding storage sites are above the one or more binding sites, and
iii. A uniform thickness of the layer, controlled by spacers and plates, of less than 250 μm and substantially less than the linear dimension of a given area of each storage site,
Is a step,
(E) After (d) and while the plate is in the closed configuration,
(1) incubating the sample for a relevant length of time and then stopping the incubation, or
(2) Samples are sampled for a time greater than or equal to the minimum relevant time length and then evaluated for binding to the binding site of each target analyte within a time less than or equal to the maximum relevant time length. Steps to do
And
The relevant length of time is
i. More than the time required for the target analyte of (a) to diffuse over the thickness of the layer of uniform thickness in the closed configuration, and
ii. Significantly less than the time required for the target analyte of (a) to diffuse laterally over the linear dimension of a given region of the binding site,
Thereby, a reaction is generated, in which in the end of the incubation in (1) or during the evaluation in (2) most of the capture agent-target analyte-detection agent sandwich bound to each binding site is corresponding. Derived from the relevant volume of the sample,
Incubation allows each target analyte to bind to the binding site and the detection agent, and the corresponding relevant volume is the portion of the sample at the corresponding storage site in the closed configuration and the edges of adjacent storage sites. The separation distance between the moieties and the edges of the adjacent binding sites is greater than the distance over which the target analyte or detection agent can diffuse in a relevant time period, and provides a fluid isolation between the binding and/or storage sites. No step
Including the method.
Embodiment 12. A device for parallel multiplexed assays of liquid samples without fluidic isolation, comprising:
Including a first plate and a second plate,
i. The plates are movable relative to each other in different configurations, one or two plates being flexible,
ii. One or two of the plates includes spacers secured to each plate, the spacers having a predetermined substantially uniform height and a predetermined constant distance between the spacers.
iii. Each said plate has on its respective surface a sample contact area for contacting a sample containing one or more target analytes capable of diffusing into the sample,
iv. The first plate has on its surface one or more binding sites each having a predetermined region containing a capture agent that binds and immobilizes a corresponding target analyte of the sample, and
v. The second plate has one or more corresponding storage sites on its surface, each storage site having a predetermined area, which upon contact with the sample dissolves in the sample and diffuses into the sample. Contains a concentration of detection agent,
Each capture agent, target analyte, and corresponding detection agent can form a capture agent-target analyte-detection agent sandwich at the binding site of the first plate,
One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates, and
Another configuration is the closed configuration, which was configured after the sample was deposited in the open configuration, and in the closed configuration,
i. At least a portion of the sample is compressed into a layer of uniform thickness, said layer contacting and constrained by the inner surfaces of the two plates, and having one or more binding sites and one or more Contact the storage site of
ii. One or more corresponding storage sites on one or more binding sites,
iii. A uniform thickness of the layer, controlled by spacers and plates, and smaller than 250 μm, substantially smaller than the linear dimension of a given area of the storage site, and
iv. There is no fluid isolation between the binding site and/or the storage site,
The separation distance between the edges of adjacent storage sites and the separation distance between the edges of adjacent binding sites is greater than the distance over which the target analyte or detection agent can diffuse within the relevant time period, and the binding sites and/or A device in which there is no fluidic isolation between storage sites.
Embodiment 13A. A system for rapidly analyzing a sample using a mobile phone,
(A) one or two plates of the CROF device are movable relative to each other in different configurations,
i. One configuration is an open configuration in which the two plates are partially or completely separated, the spacing between the plates is not adjusted by spacers and the sample adheres to one or two plates, and
ii. Another configuration is a closed configuration, which was configured after the sample was deposited in an open configuration, in which at least a portion of the sample was compressed by the two plates into a layer of uniform thickness to provide a uniform thickness. A layer of thickness, in contact with and limited by the inner surfaces of the two plates, regulated by spacers and plates, a CROF device,
(B) i. One or more cameras for detecting and/or imaging the sample,
ii. Electronic components, signal processor, hardware and software for receiving and/or processing detected signals and/or images of samples and for telecommunications
A mobile communication device including
(C) with a light source from either the mobile communication device or an external source
Including the system.
Embodiment 13B. A method for rapidly analyzing a sample using a mobile phone, comprising:
(A) depositing a sample on the CROF device of the system of Embodiment 13A,
(B) assaying the sample deposited on the CROF device to produce a result,
(C) transmitting the results from the mobile communication device to a location remote from the mobile communication device.
Including the method.
Embodiment 14.
(A) obtaining a sample containing an analyte capable of diffusing into the sample,
(B) obtaining a first plate and a second plate that are movable relative to each other in different configurations, one or two plates including spacers fixed to the respective plates. , The spacer has a predetermined uniform height, and the first plate includes an analyte assay region having a predetermined region on its surface,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, wherein the open configuration is such that the two plates are partially or completely separated and The distance between the plates is not adjusted by the spacer, a configuration step,
(D) after (c), using two plates, compressing at least a portion of the sample into a layer of uniform thickness, the uniform thickness being determined by the inner surfaces of the two plates. Limited, the uniform thickness of the layer is controlled by spacers and plates, and is substantially less than the linear dimension of a given lateral region of the analyte assay region, said compression being
Uniting the two plates, and
Applying an external force to the outer surfaces of the plates so that the plates are pressed together into a closed configuration
Wherein the force creates a pressure on a plate located above at least a portion of the sample, the pressure laterally spreading at least a portion of the sample between the inner surfaces of the plate, and the closed configuration. Is a configuration in which the spacing between the plates in the layer of uniform thickness region is adjusted by spacers, a step,
(E) while the plate is in the closed configuration, (i) for a time substantially equal to or longer than the time required for the analyte to diffuse over the thickness of the layer of uniform thickness, and (i) Incubating the sample for a time significantly shorter than the time required for the analyte to diffuse across the analyte assay area, and
(F) immediately after step (e), stopping the incubation and measuring the analyte in the assay area, or continuing the incubation while the plate is in the closed configuration and the analyte is in the analyte assay area. Measuring analytes within the assay area in a significantly shorter time than required to diffuse throughout
A method of analyzing a liquid sample, comprising:

  The following description may be applied to the above Embodiments 1 to 14.

  In any embodiment that uses CROF, the spacers are within the sample area and inside the relevant area of the sample for good uniformity of sample thickness control.

  In any embodiment that uses CROF, at least one of the two plates may be a plastic film with a thickness of 1 μm to 50 μm.

  In any embodiment that uses CROF, at least one of the two plates may be a plastic film with a thickness of 50 μm to 100 μm.

  In any embodiment that uses CROF, at least one of the two plates may be a plastic film with a thickness of 100 μm to 150 μm.

  In any of the embodiments using CROF, at least one of the two plates may be a plastic film with a thickness of 150 μm to 250 μm.

  In any embodiment using CROF, the two plates may be thin plastic films with the thickness of each plate independently selected from 10 μm to 300 μm.

  In any embodiment using CROF, the two plates may be thin plastic films with the thickness of each plate independently selected from 100 μm to 200 μm.

  In any embodiment using CROF, the two plates may be thin plastic films with the thickness of each plate independently selected from 10 μm to 100 μm.

  In any embodiment that uses CROF, the height of the spacers on the plate may range from 5 nm to 100 nm.

  In any embodiment that uses CROF, the height of the spacers on the plate may range from 100 nm to 500 nm.

  In any embodiment that uses CROF, the height of the spacers on the plate may range from 500 nm to 1 μm.

  In any embodiment using CROF, the height of the spacers on the plate may be in the range of 1 μm to 2 μm.

  In any embodiment that uses CROF, the height of the spacers on the plate may be in the range of 2 μm to 5 μm.

  In any embodiment that uses CROF, the height of the spacers on the plate may be in the range of 5 μm to 10 μm.

  In any embodiment that uses CROF, the height of the spacers on the plate may be in the range of 10 μm to 30 μm.

  In any embodiment that uses CROF, the height of the spacers on the plate may be in the range of 30 μm to 50 μm.

  In any embodiment that uses CROF, the height of the spacers on the plate may be in the range of 50 μm to 100 μm.

  In any embodiment that uses CROF, the distance between spacers (ISD) is 200 μm or less.

  In any embodiment that uses CROF, the distance between spacers (ISD) is 150 μm or less.

  In any embodiment that uses CROF, the distance between spacers (ISD) is 100 μm or less.

  In any embodiment that uses CROF, the distance (ISD) between the spacers is 80 μm or less, such as 60 μm or less, 40 μm or less, or 20 μm or less.

  In any embodiment that uses CROF, the width to height ratio of the spacers is at least 1.5 (eg, at least 2, at least 3, at least 4 or at least 5).

  In any embodiment using CROF, the ratio of column width to column height may be at least 1, at least 2, at least 5, or at least 10.

  In any of the embodiments using CROF, the distance between the plates is in the range of 2-50 μm, and any measurement has a saturation time of less than 1 minute.

  In any embodiment that uses CROF, the method comprises washing.

  In any embodiment that uses CROF, the method does not include a wash.

  In any embodiment using CROF, after incubation for less than 1 minute, the method has a sensitivity of less than 1 nM, for example at 0.1 nmol, 10 pmol, 1 pmol, 0.1 pmol, 10 fmol, 1 fmol or 0.1 fmol. is there.

  In any embodiment that uses CROF, the ratio of the period to the width of the spacer is less than about 7.0 (eg, about 7.0-1.0), especially if the column height is less than about 100 μm. May be.

  In any embodiment using CROF, the plate may have a thickness of 20-200 μm, for example 10-50 μm or 50-200 μm.

  In any embodiment that uses CROF, the sample volume may be less than 0.5 μm, such as less than 0.5 μm, less than 0.4 μm, less than 0.3 μm, less than 0.2 μm, or less than 0.1 μm. ..

  In any embodiment using CROF, the distance between the spacers may be less than 200 μm, for example 20-200 μm, 20-50 μm or 50-200 μm.

  In any embodiment that uses CROF, the device can be manually compressed in less than 1 minute, eg, less than 10 s.

30 Homogeneous Assays with Signal Amplified Surfaces In many applications of the assay, it is desirable to avoid wash steps, especially in PoC or other rapid assays. One aspect of the present invention relates to devices, systems and methods that can avoid assay washes.

  By incorporating and/or using a signal amplification surface, the disclosed devices, systems and methods can facilitate assay performance in non-washed conditions. Surface amplification surfaces can only amplify light emitted at short distances from the surface (eg, 20 nm, or 50 nm, or 100 nm). An example of the surface amplification layer is D2PA.

31 Example of Assay Acceleration Using CROF with Ring Spacer A polystyrene thin film was used as one of the CROF plates, a thin glass plate was used as the other plate, and a wax ring was used as a spacer and fixed on the polystyrene plate. It was In the CROF process, 2 uL (microliters) of sample is dropped into the ring spacer (and forms a small drop in the center when dropped) and compressed by two plates into a thinner film, The distance between was adjusted by a ring spacer (ie a closed configuration of two CROF plates). The plate was pressed by hand. The sample thickness was found to be uniform in the closed configuration of the plate. One main reason for being uniform is that the sample volume is the same as the volume between the ring spacer and the two plates. Both immunological and DNA hybridization assays were performed.

  In the immunoassay test (wax ring spacer about 40 μm in height and 0.8 cm in diameter), protein A was applied as a capture agent to a polystyrene surface and labeled IgG was used as the analyte. After incubation for binding of protein A to labeled IgG, unbound IgG was washed and captured IgG label was measured. Examine different incubation times. Our experiments showed that binding saturates within an incubation time of less than 1 min (ie below 1 min the signal of the captured IgG label does not change with incubation time). Such short saturation incubation times are expected for 40 μm intervals (and thus sample thickness), as the diffusion time of IgG in solution over a 40 μm distance is approximately a few seconds.

  The inventors have further tested the incubation of such a direct assay in a regular 96-well plate with a sample thickness of 3 mm and have found that a typical saturation incubation time is about 120 min. .. If the incubation process is limited by the diffusion of labeled IgG, the incubation time can be reduced from about 120 min to 1.28 sec by reducing the sample thickness from 3 mm to 40 μm, which is less than 1 min saturated incubation. Consistent with the observation of time.

  In the DNA hybridization test (wax ring spacer having a height of about 52 μm and a diameter of 0.7 cm), streptavidin-BSA is a molecular linking layer on a polystyrene substrate and is linked to a biotinylated capture chain, Captures the labeled target strand by hybridization. After incubation, the non-hybridized target strand is washed and the labeled signal is examined. Examine different incubation times. According to our experiments, the hybridization saturates within an incubation time of 30 sec (ie below 1 min the signal of the captured IgG label does not change with the incubation time). Such short saturation incubation times are expected for 52 μm intervals (and hence sample thickness), as the diffusion time of the target probe in solution over a 52 μm distance is approximately a few seconds. (Experimental details are disclosed, for example, in US Provisional Application No. 62/202,989).

  As a reference, the same assay with a thicker sample thickness was examined and we found that a 1 mm thick sample required approximately 20 min to reach saturation incubation.

  (Experimental details disclosed in, for example, US Provisional Application No. 62/202,989)

Example of Assay Acceleration (QAX and QMAX) Using CROF with 32 Columnar Spacers E-1.1 CROFF device for columnar spacer arrays with spacer height of 30 μm to achieve measurements with saturation incubation times less than 30 seconds. QAX assay used Check QAX by CROF and achieve saturation time of approximately 30 sec. Figure 13. a and 13. b shows this experiment. In the experiment, one of the CROF plates is pre-deposited with the capture agent and the labeled detection agent and dried before the CROF process, after which the sample is dropped on the plate and the other plate is closed by the CROF process. Dropping of the sample took a few seconds and the CROF process was completed in less than 10 seconds. According to our experiments, for a spacer height of 30 μm, the saturation incubation time is less than 30 seconds.

  Plates, Samples, Reagents (1) CROF using a self-supporting CROF device is (i) a 2.5 cm x 2.5 cm area X-plate made of 175 μm PMMA film and having a spacer array in the sample contact area. The spacer array is a rectangular grating having a constant period of 120 μm/110 μm (transverse direction of x and y), all the spacers are columnar, and the same height of 30 μm, width of 40 μm in the x direction, and width of y direction. Having the same rectangular shape with a width of 30 μm, the spacers are made of a sample material (PMMA) that is the same as the plate, and the PMMA film is manufactured by nanoimprinting in a mold (hence the spacers are defined on the plate). Fixed at a spacer height of 80 μm and a spacer interval of 80 μm), an X-plate, and (ii) a glass plate having a flat surface (thickness: 1 mm, 3 cm×5 cm). The surface of the X-plate and the glass plate are untreated and hydrophilic to the sample. (2) Prior to the sample dropping and the CROF process, a dried anti-IgG scavenger (cAb) was previously applied to the glass plate, and (3) the anti-IgG dried onto the X-plate before the sample dropping and the CROF process. Detection agent (dAb) was previously applied, and (4) the sample is human IgG in different defined concentrations of BSA buffer.

  Experimental Steps and Results A small sample containing the analyte (human IgG) is dropped onto the surface of the plate of one of the CROF devices described in E2-1. The sample on the plate initially forms a puddle, but the original puddle was adjusted by the spacer array by placing the other plate of the CROF device over the puddle and compressing the two plates together. There is a sample spread in a large area and ultra-thin sample film (about 30 μm), and spread in it. Then touch the glass plate with a human hand to press the X-plate uniformly (center-to-center) onto the droplets for 5-10 s, let go, wait 30 s, and hold the plate in its closed configuration.

  Then different samples (with different CROF instruments) are incubated, washed and measured (light signal) within different times. The result is shown in FIG. b, and FIG. It shows that the saturation incubation time of the QMA assay described in a is less than 30 seconds.

E. 1.2 QMAX Assay and Homogeneous Assay QMAX is experimentally tested using M-plates (eg D2PA) to amplify the signal. The QMAX assay is also compared to the QAX assay, which lacks the signal amplifying MOPate. Inspect for inhomogeneity (wash) and homogeneity (no wash). The test assay is a human IgG fluorescent immunoassay using QAX and QMAX.

  Materials and methods: (30 μm post height, 30 μm×40 μm post size, 80 μm ISD) 25 mm×25 mm X-plate, 25 mm×25 mm size M-plate, (application sequence) in (a ) DSU, protein A, anti-human IgG (applied to substrate plate and dried), (b) human IgG (analyte), and (c) anti-human IgG-IR800 reagent (applied to storage site of x-plate) And dry) assay reagents.

  Results (also shown in FIG. 14): Our experiments show that for a 30 μm spaced CROF device in a closed configuration, saturation incubation was within 1 min and total readings with QMAX washes. When the sensitivity is LoD=2 pM and there is no QMAX wash (heterogeneous), the total read sensitivity is LoD=10 pM and with the QAX wash the total read sensitivity is LoD=200 pM and there is no QAX wash (non-homogeneous). The total read sensitivity is LoD = (unreadable, no difference between different analyte concentrations) when homogeneous.

33 ADDITIONAL EXEMPLARY EXPERIMENTAL TESTS AND PREFERRED EMBODIMENTS This section describes additional exemplary experimental tests and observations of the present invention, and additional preferred embodiments, which share the following common observations using the following conditions: provide.

  Volume of deposited sample Unless otherwise stated, all samples deposited on the CROF plate have an unknown volume, ie the exact volume is unknown at the time of deposition.

  Plates In the CROF apparatus used in this section, unless otherwise stated, one of the two plates is called the "X-plate" and is the only plate with spacers. The other plate is called the "substrate plate" and has a flat surface and does not have any spacers. Inspect different materials for plates and spacers (including glass, PMMA (polymethacrylate), and PS (polystyrene)), different plate thickness and spacer geometry (shape and size). The sample-contacting surface of each plate is a flat surface (excluding protruding spacers), which generally has a surface smoothness variation of less than 30 nm, but many flat surfaces have plate flexibility, an inherent surface flatness. Surface flatness variation due to the degree (not related to the flexibility of the plate), or both. Some plates have internal surface smoothness variations greater than 30 nm. Unless otherwise stated, the plates used in the examples have a typical dimension of at least 25 mm wide and at least 25 mm long.

  Spacers Unless otherwise stated, all spacers in this section are (i) fixed to the sample surface of the X-plate and by embossing the surface (so that the spacer material is the same as the X-plate). Manufactured, (ii) pillars with rounded corners, approximately straight sidewalls with a tilt angle of less than 5 degrees from the normal, a flat top surface, and a rectangular or square approximately uniform cross-section with uniform spacer height. An array and (iii) the distance (ISD) between the inner spacers fixed in the X and Y directions, respectively (note that the spacing in the X direction may differ from the spacing in the Y direction) (FIG. 17. b)). Further, the lateral shape of the columnar spacer is a square or a rectangle with rounded corners, and the height, size, distance between the spacers, shape and material of different spacers are inspected.

  Manufacturing of spacers X-Manufacturing spacers embossed on the plate surface by nanoimprinting, pressing the die directly on the plate, and embossing the originally completely flat surface with a flat spacer protruding from the surface. To do. In embossing in which the plastic material is flowable, a temperature above the glass transition temperature of the plastic material is used. The mold is manufactured by lithograph and etching, and in some cases by electroplating the master model. The mold is made of silicon, silicon dioxide, or nickel.

  FIG. 17 shows an example of spacers manufactured on a plate. The spacer is manufactured by directly embossing the surface of the plastic plate using a mold. 17(a) and 17(b) are top views of optical micrographs of square spacer gratings. (A) A columnar spacer size is 46 μm×46 μm and the distance between columns is 54 μm, and (b) A columnar spacer size is 10 μm×70 μm and the distance between columns is 10 μm. Yes, (c) Columnar spacer size is 30 μm×40 μm and spacer height is 2 μm, and (d) Columnar spacer size is 30 μm×40 μm and spacer height is 30 μm It is SEM. The micrographs show that (1) the tops of the columnar spacers are very flat, (2) the spacers have a substantially uniform cross-section, (3) the columnar spacers have rounded corners, and a radius of curvature of about 1 μm. It has. A large radius of curvature (eg, a small sharp edge) is preferred, as sharp edges lyse cells or affect fluid flow as compared to circular edges.

  Using a profilometer, we measure the height of the pillars in the 2 cm x 2 cm area of the X-plate. The general uniformity of the heights of the columnar spacers of the X-plate manufactured by the above method is as follows: average variation values of 4 nm, 10 nm, 100 nm and 0 for spacer heights of 2 μm, 5 μm, and 30 μm, respectively. Relative mean variation values of 0.2%, 0.2%, and 0.33%.

  General Experimental Procedure As shown in FIG. 15, first, a small volume (several μL or less) of a sample forming a small pool was attached to a substrate or an X-plate. Second, the plates were brought together so that the sample surfaces of the plates overlap. Third, the hands are used to press the plate into a closed configuration where the sample is a thin film with a larger area than the original pool. Fourth, related to the hand. Fifth, it performs different measurements in the closed configuration. Details of the steps are shown below.

  Sample attachment method Two types of sample attachment methods are used. In one method, the sample is attached to the plate with a pipette. In another method, the blood sample is deposited directly from the subject's finger (taken with the instrument) by contacting the subject's blood with the plate. Do not dilute blood that has adhered directly to the plate from your fingers. We have found in our experiments that the final experimental results are independent of the sample attachment method unless otherwise stated.

  Sample deposition is done in the room under standard room conditions without the use of any special temperature control or dust filtration. Under such conditions, the present inventors have found that (1) the flexible plate used in conformity with the dust has the sample thickness of other regions adjusted by the spacer and self-holding of the sample thickness. And (2) the area with dust is a very small part of the total available area and the measurement is done in an area unaffected by dust, so It was discovered that dust that did not affect the final measurement results. The selection of the dust free area is completed by the optical image.

  In some cases, the two plates have a surface protective cover to reduce the number of dust particles falling on the plates. In some cases, two plates are placed with the inner surface of the sample to prevent dust and other contamination.

  Surface Wettability of Plates We measured the surface wetting properties of the different plates used in the exemplary experiments. The table below shows the measured contact angles of 5 μL of the sample, which were untreated or after treatment with different plate materials (glass, PMMA and PS) and different surface topography (flat surface and X-plate sample surface). Located on the surface with different sample formats (water, PBS buffer (phosphate buffered saline) and blood), the X-plate is 175 μm thick PMMA and the sample surface is 2 μm high, 30 μm It has a columnar array (that is, spacers) with a lateral size of ×40 μm and a period of 110 μm/120 μm (that is, the distance between spacers is 80).

  Experiments show that (1) untreated surfaces of glass, PMMA, and PS all have hydrophilic surfaces (ie, contact angles less than 90 degrees), and (2) untreated glass surfaces are untreated. Of PMMA and PS (greater hydrophilicity), and (3) the contact angles of water, PBS and blood are similar, blood has slightly better wettability than water and PBS. However, (4) the untreated PMMA X-plate has about the same sample contact angle as the untreated PMMA plate, and (5) as expected, the surface wetting properties show that the surface treatment makes it more hydrophilic or It has been shown that it can vary significantly to become hydrophobic.

  The surface hydrophobicity of the plate can be changed by surface treatment. For example, for PMMA X-plates, improve the hydrophilicity by exposing the surface in oxygen plasma and treat the surface with tridecafluoro-1,1,2,2-tetrahydrooctyltrichlorosilane. Improves the hydrophobicity. For water, PBS buffer and blood samples, the contact angles are 25, 27 and 28 degrees for the X-plate after hydrophobic treatment and 105, 104 for the X-plate after hydrophilic treatment, respectively. And 103 degrees.

  In the following description, all sample surfaces of the plate (ie, the inner surface in contact with the sample) are untreated unless otherwise stated.

  Area and Height of Attached Sample When the water sample is attached to the plate in an open configuration with a pipette, measure the area and height of the sample on the plate.

  Experiments show that a typical sample attached to the plate in the open configuration has a much greater thickness than the closed configuration.

  We have found that in the closed configuration of the plate, (1) the total sample area extends from a diameter of a few millimeters to a few centimeters (depending on the spacer height), and (2) the spacer array is square. It was observed that with the grid, the area of the sample in the closed configuration of the plate was also approximately square, with the edges of the sample aligned with the square grid of the spacer. Thus, it has been shown that different spacing of spacers can control the final sample area under the closed configuration. If the spacer has a rectangular grid, the final sample area in the closed configuration should be rectangular. If the spacer has a radial circular pattern, the final sample area in the closed configuration should be circular.

  Hand Press In all experiments in section 30, the plates in the CROF process are brought together and manually compressed into the closed configuration of the plates. A wide area of the CROF plate is pressed last to obtain a uniform sample thickness, generally one area of the thumb is pressed and different areas of the CROF plate are rubbed. The process of manually pressing the CROF device (plate) into the closed configuration is called "hand pressing".

  Self-holding observations show that after pressing the CROF plate to the final state and releasing the compressive force (eg pressing hands), the sample thickness between the two plates is still the spacer height, unless otherwise noted. And is kept at a constant thickness for an extended period of time (until the sample is finally dried). This observation is called "self-holding". Self-holding is a capillary force between the plate, liquid sample and environment (eg air) caused by surface tension. Observe that hand pressing and self-holding of the CROF device gives good sample thickness, as shown in E-1.

により計算されることができ、ここで、ｃは光速であり、Δｖは周波数領域における期間であり、ｎはプレート間の試料の屈折率である。 Measurement of plate spacing and sample thickness In all experiments, the distance between the inner surfaces of two plates in a closed configuration (ie, the sample surface) is the Fabry-Perot cavity resonance (FP resonance) caused by the inner surfaces of the plates. ) Is measured by. Due to the difference in optical index between the sample (and air) and the inner surface, each inner surface of the plate acts as a light reflector and the two inner surfaces form an optical resonator. The FP resonance spectrum is periodic, and the inner surface spacing h (and therefore sample thickness) at the optical measurement point is

Where c is the speed of light, Δv is the period in the frequency domain, and n is the index of refraction of the sample between the plates.

  In the FP resonance examination by the present inventors, the light source has an area of about 2 μm×2 μm. Generally, we measure the spacing of the inner surface of the plate at 25 different points within a 1.6 cm x 1.6 cm area around the center of the CROF device, where 25 points are 4 mm. It is a 5×5 square grid with a period (ie the distance between two adjacent points). It does not measure the area occupied by the spacers (ie the columns).

  Since the inner surface contacts the sample in the closed configuration of the plate, the measured inner surface spacing is the same as the sample thickness at the measurement point.

を用いて計算される。 Average sample thickness, H The average sample thickness H is the plate spacing and formula measured at 25 points.

Is calculated using.

The deviation of the thickness of the sample refers to the deviation of the average thickness H of the sample on the predetermined region of the height H ₀ of the predetermined spacer. _(H-H 0). The relative sample thickness deviation refers to the deviation divided by the height of a given spacer: [(H−H ₀ )/H ₀ ]. A positive thickness deviation means that the sample is thicker than the spacer height on average, and a negative thickness deviation means that the sample is thinner than the spacer height on average.

Sample Thickness Uniformity Sample thickness uniformity over a given area is defined as the standard deviation of sample thickness over a given area.

32.1 Sample Thickness Deviation and Uniformity in Hand Press Self-Supporting CROF Experimentally, we found that in a CROF device and process, the sample thickness deviation and uniformity of the closed configuration of the plate after the hands were released. Study parameters that can affect sex. The parameters are the distance between the spacers (IDS), the shape and size of the spacer (eg, the lateral dimension of the spacer, the height of the spacer, the ratio of the width to the height of the spacer, the spacer region filling factor (the ratio of the total area to the spacer area). Or the ratio of the spacer period to the width), the mechanical strength of the spacer and plate material (Young's modulus), the thickness of the plate, and the surface flatness of each plate, but not limited thereto. Some experimental findings and preferred embodiments are presented: Spacer height, IDS, period and spacer lateral size definitions are shown in FIG.

  E-1.1 Effect of IDS (distance between spacers), plate thickness, and material on sample thickness Experimentally, we find that the distance between spacers (ISD) of a periodic spacer array is It was observed that sample thickness deviations (from spacer height) and uniformity in the closed configuration in the CROF preloss can be significantly affected.

  FIG. 18 shows the effect of IDS, plate thickness and material on sample thickness. FIG. 7 shows measurements of sample thickness deviation and uniformity for different plate and spacer materials, different plate thickness and distance between spacers (IDS) for different samples. The spacers are periodic arrays and have spacer heights of 5 μm, flat tops, square shapes (10×10 μm post lateral size, nearly uniform cross section, and rounded corners). The IDS was 20 μm, 50 μm, 100 μm, 200 μm and 500 μm, respectively. The substrate was an untreated 250 μm thick flat surface (1″×1″ area) PMMA plate. The X-plates on which the spacers were directly manufactured were untreated 175 μm and 50 μm thick PMMA plates and untreated 125 μm and 25 μm thick PS, respectively. The samples were 2 μL of blood (dropped in direct contact with the finger), saliva or PBS (dropped with a pipette), respectively. The CROF device was manually pressed and rubbed in a 1 inch x 1 inch area and self-held after pressing. Sample thickness was measured in a closed configuration of the CROF device.

FIG. 18 shows that for a given experimental condition and for square shaped spacers (10×10 μm post lateral size, nearly uniform cross section, and rounded corners):
(1) When the ISD is 20 μm, 50 μm, 100 μm, the average final sample thickness is 5.1 μm to 5.2 μm, which is very close to the predetermined spacer height of 5 μm and less than 4%. Deviation and uniformity (ie, deviation and uniformity may be less than 4% if ISD is about 120 μm or less).
(2) However, when the ISD is 200 μm and 50 μm, the average final sample thicknesses are 4.3 μm and 3.5 μm, respectively, which are clearly smaller than the predetermined spacer height (5 μm), and − It has thickness deviations of 13.9% and -30.9% and uniformity of 10.9% and 27.7%, respectively. This shows that when the ISD is larger than about 200 μm, not only the average value of the thickness is significantly decreased, but also the uniformity is very low.

  For a columnar spacer array with lateral dimensions of 40 μm×40 μm (FIG. 18), if the ISD is 60 μm, 150 μm, 100 μm, the average final sample thickness is 5.1 μm-5.2 μm, and the spacer with a predetermined 5 μm Very close to the height of and of less than 4% thickness deviation and uniformity (ie, deviation and uniformity may be less than 4% if the ISD is about 100 μm or less).

E-1.2 Effect of IDS/(Eb^3) on Sample Thickness According to our experiments (as shown in FIG. 19), small sample thickness deviations and good uniformity are achieved. For that, the SD ⁴ /(h ^x E) of the X-plate (x=1 in the plot) should be less than 10^6 μm^3/GPa, ISD is the distance between the spacers and h is the material Height (thickness) and E is the Young's modulus of the material.

  In all methods and devices using CROF, in some embodiments, the value of SD4/(hxE) (x=1 in the plot) is less than 10^6 μm^3/GPa, less than 5x10^5, 1x It is less than 10^6, less than 5x10^6, and so on.

  In any embodiment, the flexible plate has a thickness in the range of 20 μm to 250 μm (eg, in the range of 50 μm to 150 μm) and a Young's modulus in the range 0.1 to 5 GPa (eg, 0.5 to Within the range of 2 GPa).

  In any embodiment, the product of the flexible plate thickness and the flexible plate Young's modulus may be in the range of 60 to 750 GPa-μm.

E-1.3 Effect of Spacer Size and Height on Sample Thickness According to our experiments (eg FIG. 20), in order to achieve small sample thickness deviations, given plate thickness, For samples and presses, the IDS should be about 150 μm or less.

E-1.4 Effect of Width to Height Ratio of Spacer on Sample Thickness According to our experiments (eg, FIG. 21), in order to achieve small sample thickness deviations, for a given plate, For thickness, sample and press, the ISD is between 20 μm and 150 μm, the width to height ratio (WRH) of the pillar is greater than 1, and in some embodiments, preferably 2 or more.

  This means that when the WHR is about 1 or more, the spacer is strong enough to maintain the hand press pressure and rubbing, otherwise, for all ISDs, both deviation and uniformity are Indicates low and large.

E-1.5 Effect of Spacer Filling Factor on Sample Thickness According to our experiments (eg, FIG. 22), a small sample thickness deviation and good thickness uniformity were determined to be achieved. For the plate thickness, sample, the spacer fill factor should be about 2.3 or more.

For example, the deviation and uniformity of less than 4% achieved in FIG. 22 means that for a given pillar area and IDS, and for a given spacer area fill factor (eg, the ratio of column cross section to total area), the PS column The body is strong enough to support the pressure and rubbing of a hand press. The deformation of the PS column is such that the thumb pressure is about 1 to 10 kg/cm ² (10^5 Pa), the PS Young's modulus is about 3 GPa, and the columnar spacer filling rate of 20 μm width and 100 μm ISD is about 4. %, so that the relative deformation (pressure) in the thumb press of the cylinder is between 1% and 0.1%, which is in agreement with our experimental observations. To do.

E-1.6 Effect of Plate Thickness on Sample Thickness According to experiments by the present inventors (eg, FIG. 23), (i) small sample thickness deviation (5% or less) and good thickness uniformity To achieve a given plate thickness, for a sample, at least one plate should have a plate thickness of less than 200 μm, and (ii) the X-plate and the substrate are both greater than 200 μm. If too thick, they are too hard to overcome dust and lead to lower spacer uniformity/deviation.

E-1.7 Effect of Substrate Plate on Sample Thickness According to our experiments (eg FIG. 25), using a thick (1 mm) glass substrate plate resulted in smaller sample thickness deviations and better thickness. The maximum IDS for uniformity can be extended from 150 μm to 200 μm for PMMA substrates.

E-1.8 Effect of Modifying Plate Surface Wetting Properties on Self-Retention According to our experiments (eg, FIG. 24), (1) good self-retention of the CROF device was due to the two inner surfaces of the CROF device. At least one needs to be hydrophilic. (2) When both two inner surfaces of the CROF device are hydrophilic, it provides the best self-holding and sample thickness control and uniformity. (3) If one inner surface of the CROF device is hydrophilic and the other inner surface is hydrophobic, the sample area needs to be larger than 0.5 cm ^{2 in} order to obtain good self-holding. (4) When both of the two inner surfaces are hydrophobic, self-holding is insufficient or unsuccessful (unstable). (The lines in the figure are for guiding the eyes.)

E-1.9 Effect of hand pressing time on sample thickness Our experiments have found that the CROF device can self-hold for a pressing time of 1 s to 60 s and has similar good performance. Without pressing anything (pressing for 0s), the CROF device has poor performance and cannot self-hold.

E-1.10. Comparison of the effect of periodic column spacers and random ball spacers on sample thickness shows that the measurement results in FIG. 27 show that a CROF device with periodic column spacers is much better than a random ball (eg bead) spacer under given experimental conditions. It is shown to have small sample thickness deviations and higher homogeneity (both less than 5%). Specifically, for 20 μm, 50 μm and 100 μm ISDs, the average thickness deviation and uniformity of the columnar spacers of periodic and uniform cross section are 2.3% and about 3.4%. However, when using the random ball spacers with the average ISD of 20 μm, 50 μm, and 100 μm, the average thickness deviation and uniformity using the glass cover plate with the thickness of 220 μm are 11.2% and 12.5%. The sample thickness deviation and uniformity using PMMA cover plate of 175 μm is 10.8% and 20%, with sample thickness deviation of more than about 5 times and lower uniformity.

E1.12. Other Findings FIG. 28 shows the effect of different X-plate and substrate thicknesses on sample thickness.

  Our experiments have found that liquids pipetted directly from the finger have similar performance in final sample thickness and uniformity.

  Our experiments have also found that the liquid dripped onto the substrate or X-plate has similar performance in measured sample thickness and uniformity.

32.2 Complete blood count E2.1 of undiluted whole blood using self-supporting CROF CROF instrument used The CROF instrument used in all the tests of Example 32.2 was an X-plate and a flat glass plate. Including. The X-plate is PMMA with an area of 2.5 cm×2.5 cm and a thickness of 175 μm, and has a periodic spacer array in the sample contact area. The glass plate is a flat surface with a thickness of 1 mm and an area of 3 cm×5 cm. The spacers on the X-plate are embossed directly into the initially flat PMMA film, thereby being made of PMMA (the same material as the X-plate) and in contact with the X-plate.

  Each spacer is columnar with a substantially uniform cross section, a flat top, and a rectangle with widths of 40 and 30 um in the x and y transverse directions, respectively. All spacers on a given X-plate have the same spacer height. The periodic spacer array has a rectangular grating with a constant period of 120 um and 110 um (x and y respectively) with a given constant spacer spacing (ISD) of 80 μm.

  The surfaces of the X-plate and the glass plate are untreated and hydrophilic to human blood dripping on the surface with a contact angle of about 40-50 degrees. Both plates are transparent to visible light.

E2.2 Sample, Manufacturing, Adhesion, CROF Process, Self-Retention Unless otherwise stated, all blood samples were from healthy subjects, fresh, and directly attached to the CROF plate, undiluted and anticoagulated. No agent was added.

  In all experiments in Example 3, unless otherwise noted, blood was taken from a human finger and blood was deposited on the CROF plate by direct contact of the blood with the plate. Direct contact typically deposits about 0.1-1 μL of blood on the surface of the plate. Less than about 60 seconds after blood deposition, the CROF process was applied to compress the blood sample into a thin film and then the measurement was performed. No anticoagulants or liquid diluents were added to blood samples unless otherwise noted.

In certain experiments requiring WBC staining, the reagent, a dried acridine orange dye layer, was pre-applied to one inner surface (sample contacting surface) of the plate of the CROF instrument prior to the blood test. The application of the dry dye layer is carried out in the following order: (a) adding 30 μL of acridine orange dye to a glass plate in water having a concentration of 20 μg/mL, (b) spreading it in an area of about 1 cm ² , (c) ) Drying for about 1 hour.

  Regardless of the method of depositing a volume of about 1 μL or less of blood on one of the CROF plates, the blood deposited on the plate formed blood bodies with a diameter of a few millimeters or less. Next, the two plates of the CROF device are closed by hand and pressed by hand for a few seconds to compress the original blood body with the two plates to produce a large thin film of blood (with lateral size approximately 1 to 3 cm). In all of Example 3, the inventors have shown that, unless otherwise noted, the hand-pressed CROF device had a uniform sample thickness adjusted by the spacer and a uniform sample thickness after the hand was released. Was able to self-hold. The sample was deposited under normal room conditions. The inventors have found that dust does not affect achieving a given final sample thickness over a large sample area. We also found that the final blood sample was caused by a rectangular grid of periodic spacers and spread into a rectangular shape with rounded corners. This step is shown in FIG.

  For samples with dry dye on the plate, the sample waited 30 seconds before any measurements.

  Blood samples deposited directly on the plate were not diluted with liquid (ie, no liquid dilution) and were mixed only with the dry reagent applied to the plate.

  All spacers have a rectangular shape with the same spacer height of 2 μm.

  The spacer height of 2 μm is selected to give a final blood sample spacing of approximately 2 μm in a closed configuration of the CROF device, which is approximately equal to the thickness of red blood cells (RBC) (2-2.5 μm). Much smaller than the diameter of RBC (6.2-8.2 μm). Such final sample thickness is such that in the closed configuration of the CROF, the RBCs are well separated from each other, there is no overlap or stacking between different RBCs, and the RBCs are accurately counted by imaging the sample area. To let

E2.3 Blood Cell Imaging Unless otherwise noted, the blood sample of Example 3 was imaged using a sample between two CROF plates in a closed configuration and using a commercial DSLR camera (Nikon) and iPhone, respectively. went. The results for each type of camera are similar. Unless otherwise noted, the images are top views of the sample through one of the plates that is transparent (ie, a two-dimensional image of the sample on a plane parallel to the surface of the plate).

  Nikon camera The sample consisted of two filters (470±20 nm bandpass filter as excitation filter and 500 nm longpass filter as emission filter), one light source (xenon lamp) and one magnification/focus lens set. Observed by a conventional commercial DSLR camera (Nikon). In brightfield mode, it is a broadband white light source that does not use any filters. In fluorescence mode, a 470±20 nm filter is placed in front of a xenon lamp to create a narrow band excitation source with a wavelength of 470 nm and a 500 nm long pass filter to block the light with wavelengths below 500 nm entering the camera. I put it in front of.

  Mobile phone The iPhone 6 was used for the experiments by the inventors.

E2.4 Effect of spacer height (sample thickness) on blood cell and red blood cell counts In our experiments, the sample thickness was controlled to be the same as the spacer height. We experimentally investigated the effect of spacer height (and thus sample thickness) on blood cells in the CROF process, and their effect on imaging and counting. The CROF equipment and process used, as well as blood deposition, are described at the beginning of this section of Example 2. Blood samples were from the same healthy subject. In one of our experiments, four different spacer heights (1 μm, 2 μm, 3 μm, and 5 μm) were examined.

  FIG. 29 shows a top view of an optical micrograph (bright field optical microscope) of a blood sample that has been CROFed in four different CROF devices, each CROF device being a rectangular grid of periodic spacers and 1 μm (a). It has different constant spacer heights of 2 μm (b), 3 μm (c) and 5 μm (d). The blood sample was deposited directly on the plate of the CROF device by the subject's finger and no anticoagulant or liquid diluent was added to the blood.

  In bright field light microscopy, RBC cells are much easier to see than WBCs. Red blood cells (RBCs), also called red blood cells, have a disc diameter of about 6.2-8.2 μm and a thickest point of 2-2.5 μm (near the edge of the disc), and a minimum of 0.8-1 μm central location. Having a thickness of.

  Observation by our optical microscope showed that for a spacer height of 1 μm, about 99% of RBCs were dissolved. For example, FIG. 29A shows that only RBC remains in the observation area. The 1 μm spacer height is significantly smaller than the average RBC thickness. The experiment demonstrated that the CROF device and process can be used to lyse cells by making the final plate spacers (by controlling the height of the spacers) smaller than the smallest dimension of the cells.

  Observation by the inventors' optical microscope (for example, FIG. 29) shows that for a spacer height (sample thickness) of 2 μm, the RBCs are all separated from each other and there is substantially no overlap between them, and It was shown to have an almost round and symmetrical shape. The distance between each RBC is clearly indicated by the perfect circular dark border of each RBC in the 2D microscope image (ie, the border completely circulates each (and only one) cell). Furthermore, microscopic observation shows that the center of the RBC is darker than the center of the edge at the center of the cell, and at a spacer height of 2 μm (sample thickness), the center of the RBC is still thinner than the edge. Indicated.

  The observation by the inventors of the present invention with an optical microscope (for example, FIG. 29) shows that when the height of the spacer (and thus the sample thickness) is 3 μm, the height of the spacer of the image of the blood sample is 2 μm. (1) RBCs have significant overlap and do not have a perfect circular dark border separating each cell so that most RBCs lie at the height of the 2 μm spacer. , Rather, some RBCs share a single dark border that is not a long circle, and (2) some RBCs appear almost circular at the spacer height of 2 μm and not more elliptical. The center dark disk of each RBC, which is transparent with a spacer height of 2 μm, is less visible. At spacer heights of 5 μm, RBCs had more overlap, more red blood cells had a non-circular shape, with almost invisible dark centers (FIG. 29).

  It is well known that RBCs desire to overlap one another in blood that is not space limited (eg, including stacking). By limiting the height of the spacers and the blood sample between the two flat plates with a spacing of 2 μm using the plate, the blood thickness is approximately at the thickest point of the RBC (which is 2-2.5 μm). Equally, therefore, at a given position on the plate surface, by limitation, only one RBC exits between the two plates and orients the RBC so as to orient the disc parallel to the plate surface, and the top view optics When observed using microscopy, it gives a good separation between the RBC, a perfectly circular dark border and a nearly round shape.

  As the spacer height and thus the sample thickness increases, such as the spacer heights of 3 μm and 5 μm, the sample thickness allows more than one RBC between the two plates at the position of the plate, and the RBC overlap and Results in the disappearance of a distinct boundary for each RBC, causing the RBC to rotate between two plates and from a disc parallel to the plate surface position, causing the top view image of the RBC to assume a non-circular shape ..

  In order to count the number of RBCs (for example used for RBC concentration measurement), it is preferable to use a sample thickness of 2 μm (for example, a spacer height of 2 μm) rather than sample thicknesses of 3 μm and 5 μm. Experimentally clearly shows that is also easy and accurate.

  The spacer height (hence the distance between the two plates and the blood sample thickness) is approximately equal to the thickness of red blood cells (RBC) (2-2.5 μm) by making it about 2 μm, but RBC( (6.2-8.2 μm) in diameter, blood cell counts are much easier and more accurate than larger sample thicknesses.

  If the final sample thickness is (2-2.5 μm), or 1.9 μm-2.2 μm, then the CROF is placed in a closed configuration, each RBC is well separated from each other, and there is no overlap between different RBCs. Alternatively, allow RBCs to be accurately counted by taking an image of the sample area without stacking.

  On the other hand, we observed that with a CROF device with a spacer height of 1 μm, most of the RBCs were lysed, but not WBCs or platelets. In our experiments, optical imaging from the top of the CROF (ie, the CROF plate is approximately parallel to the imaging plane of the microscope or camera) showed (a) the number of cells in the region, and ( b) Determine the lateral size of the area. The lateral size of the CROF device can be determined by pre-calibration. Alternatively, the lateral size of the spacer can be used as a marker to determine the lateral size of the area of the CROF device during the imaging period. Both were used in our experiments.

  In the experiment of Example 2, for a given CROF plate, the spacing between the two CROF plates (and thus blood sample thickness) was the same within the spacer height and within 5%. This sample thickness information, together with the lateral size of a given area determined from optical imaging, determines the sample volume associated with a given area, which is the lateral area of the sample times the sample. Equal to the thickness. By knowing the sample volume and the number of cells within the volume (determined by imaging), we were able to determine the concentration of cells within that sample volume.

  Figure 29b shows the ratio of (b) red blood cell area (measured from a 2D top view) to the total lateral area of the CROF plate. It is greatest when the plate spacing (ie sample thickness) is 2 μm, because some RBCs dissolve below 2 μm and RBCs overlap and rotate above 2 μm. This reduces the RBC area in the 2D image.

  One conclusion from the following experiments is that the optimized spacing size of the hemocytometer (RBC and WBC) CROF instrument is 1.9 μm to 2.2 μm, or 2 μm to 2.2 μm, or 2 μm to 2.1 μm. It means that

Another experimental finding of the inventors is that a CROF device with a 1 μm spacing between two plates lyses most red blood cells but not WBC: CROF device It has been found that if the gap spacing of the CROF device is much smaller than the RBC thickness (eg 1 μm distance between plates), the RBC will dissolve. It is still observed that WBCs are more elastic and most of them may not be dissolved.

E3.5 RBC (Red Blood Cell) Counts In one embodiment, RBCs were counted in brightfield mode without filters. Photographs were taken at 4 times, 10 times, 20 times, and 40 times magnification. Since the gap spacing (t) and field of view (A) of each magnification X-plate are known (spacers and their duration were used as scale markers (ie rulers)), the RBC concentration in the blood sample was calculated. It For example, for counting N RBCs within one field of view, the RBC concentration (C) in blood is C=N/t/A. This calculation method is similarly applied to WBC and PLT concentration measurement.

E2.6. WBC and Platelet Counts Each white cell (WBC) is also called a white blood cell or white blood cell and has a typical disc diameter of about 10-15 μm. Typical platelets (PLT) have a typical size of 1-2 μm. WBCs and PLTs have no visible dye on their own and are therefore less visible than RBCs under normal microscopy. In one embodiment, WBCs and PTLs are stained with acridine orange (AO) dye to make them more visible in counting WBCs and PTLs.

  Acridine orange is a stable dye with a natural affinity for nucleic acids. When bound to DNA, AO is intercalated with DNA as a monomer and produces strong green fluorescence upon blue excitation (470 nm excitation for WBC, 525 nm green emission). Upon binding to RNA and protein, acridine orange forms an electrostatic complex in the form of a polymer that upon blue excitation produces red fluorescence (470 nm excitation for WBC, PLT, 685 nm red emission). RBCs do not have nucleic acids and therefore cannot stain. WBCs have a nucleus, have both DNA and RNA and are therefore strongly stained. PLT has a small amount of RNA and is therefore weakly stained. FIG. 33 is shown.

  WBCs were counted in the fluorescence mode of the excitation filter at 470±20 nm, and the emission filter was a 500 nm long-pass filter, with magnifications of 4×, 10×, 20×, or 40× selected for imaging. WBCs and PLTs were counted appropriately using these embodiments.

E2.7 Measurement of different WBCs WBCs can be divided into five major subclasses: neutrophils, eosinophils, basophils, lymphocytes, and monocytes, or sometimes granulocytes, lymphocytes, and monocytes. Can be classified into three main classes. The concentration of each class in a subject's blood may have clinical significance, as different infections by viruses, bacteria, or fungi, or allergies can alter the concentration of a particular WBC subclass concentration. ..

  WBCs are nucleated that distinguish them from nucleated red blood cells and platelets. Furthermore, different subclasses of WBC have different ratios of DNA to RNA and proteins, so that DNA and RNA can be stained separately by distinguishing them with an appropriate dye.

  For example, the AO dye is intercalated with DNA as a monomer and produces strong green fluorescence upon blue excitation (470 nm excitation for WBC, 525 nm green emission). Upon binding to RNA and protein, acridine orange forms an electrostatic complex in the form of a polymer that upon blue excitation produces red fluorescence (470 nm excitation for WBC, PLT, 685 nm red emission). Therefore, different WBCs have different R/G color ratios (green emission and red emission) after being dyed with AO dye.

  The AO dye can distinguish three WBCs of granulocytes (including neutrophils, eosinophils, basophils), lymphocytes, and monocytes. Furthermore, we can directly use the camera (or iPphone) built into the RGB filter set to distinguish the green and red emission of G and R channels from one picture. Therefore, we need to use two separate filter sets (525 nm and 685 nm bandpass filters).

  As shown in FIG. 34, a total of 594 white blood cells are counted and plotted. We can clearly see that the cells cluster in three different areas corresponding to the three major leukocyte subpopulations, the shaded areas provided as a guide for the eye. The percentages for each subpopulation are shown in the table and are in good agreement with blood values in normal humans.

E2.8. Measurement of hematocrit value The hematocrit value (Ht or HCT), also called blood cell volume (PCV) or red blood cell volume fraction (EVF), is the volume percentage (%) of red cells in blood. In the X-CBC setting, we use a 2 μm gap spacing that tightly packs all RBCs with the substrate and X-plate. Therefore, the HCT in this case is equal to the RBC volume relative to the total blood volume.

E2.10. WBC Staining Speed with Dry AO Dye After 30 s, 10 min, 30 min, 90 min, WBC was dyed with dry AO dye. In the CROF process with dry AO dye on the plate surface, the AO dye can stain WBC completely in less than 1 min and does not affect other areas over time. Also, the bound AO fluoresces stronger than the unbound dye, so no washing step was required.

E2.11. Other non-fluorescent dyes that stain WBC Non-fluorescent dyes that stain WBC can simplify the WBC counting step. Crystal Violet or Gentian Violet (also referred to as Methyl Violet 10B or Hexamethylparachloroaniline chloride) is a triarylmethane dye that can be used to stain the nuclei of WBCs. Similar to AO dye, 1 mg/mL, 30 μL of acridine orange dye was dried in water for 1 hour on a glass slide with an area of 1 cm ² . Then the X-CBC experimental process is repeated. WBC stains purple. One drawback of such a method is that it is difficult to distinguish WBC subpopulations.

E2.12. CROP Anticoagulant-Free Blood Tests One advantage of the present invention is that, as observed experimentally, it is not necessary to use anticoagulants to aid counting. In our experiments, blood samples on X-plates with a blood area of 1 cm x 1 cm were examined in a CROF device with intervals of 2 μm, 3 μm, and 10 μm. Photographs of the sample at five typical points from the center to the edge were taken every 10 min for a period of 0 min to 80 min. All tested samples do not contain anticoagulants. It was observed that for a given experimental condition, the blood sample did not aggregate in the closed configuration during the observation period. This is because (1) CROFs with about 2 μm spacing (sample thickness in closed configuration) separate blood cells from each other, and (2) CROF plates protect most of the blood cells from oxygen.

Further Experiments in Blood Counts Using the E2.13 CROF and iPhone In another experiment, we found that an individual could use her/his own smartphone with less than one drop of blood (<1 μL) in less than 20 seconds. Inspect and verify the technology and small, easy-to-use equipment that allows you to perform blood cell counts in. What a person needs to do is to bring a small amount (any arbitrary unknown amount) of blood from a pricked finger into contact with the card, close the card and shoot with a smartphone.

  One aspect of the present invention is the accurate counting of blood droplets in red blood cells with only one thickness of red blood cells (approximately 2 um) and in a uniform blood layer confined between two plates. Is an observation that offers unprecedented advantages in. The advantage is that (i) the fresh undiluted whole blood without the addition of anticoagulant, the blood cells are well separated from each other, do not clot and can be easily recognized by imaging, and (ii) the sample Evaporation (in the examination area) is almost zero, and the blood cell concentration is kept constant for a long period of time. A second important technology developed by the inventors is to use a CROF card (hand-operated, foldable, disposable, stamp size (1 inch wide, paper-like thin) plastic film). Perform a blood reconstitution, measure the thickness (and thereby the volume) of the reconstituted blood sample, and mix the pre-applied dry reagent with the blood (if necessary) (all functions once It takes less than 5 seconds to complete) and is called "compression controlled open flow" (CROF). The two recent technologies reported here are a small matchbox-sized optical adapter for smartphone imaging and software for controlling the smartphone to analyze images. The smartphone method ("hemocyte counting using CROF and imaging" or BCI) is validated in comparison with standard commercial machines, commercial manual hemocytometers and microscope imaging (instead of smartphones). Forty-two or more tests using two types of blood (freshly stored in the subject) were performed for each method, including red blood cells (RBC), white blood cells (WBC), platelets, three WBC differences, hematocrit (HCT) and Mean red blood cell volume (MCV) is measured. By verification, BCI with a smartphone has the same or better accuracy as a commercially available manual hemacytometer (which can be further improved) and has the same daily stability as commercially available devices. Clearly, BCI technology has wide and important applications in cell imaging, immunological assays, nucleic acid assays, personal health monitoring, and other biochemical detections.

  The BCI device is a disposable postage stamp size plastic CROF card (1" x 1" area, thin like paper), a smartphone, and a matchbox size optical adapter (1.5 x 1.5" x 0. It includes three 7-inch (L×W×H) hardware components and software to control the smartphone, create a user interface, and analyze blood cells. All of them (except smartphones) are designed and developed by the inventor. An optical adapter (“adapter”) that includes a lens, a mirror, and a filter is attached to a smartphone, with the smartphone's flashlight and camera as the light source and inspection imager, respectively. The optical adapter also has a slot (FIG. 30) for sliding the CROF card into the proper position in front of the camera. In our current examination, we use iPhone-6.

  In the blood test using BCI (Fig. 30), first, one person pierces a finger, and then touches the card to touch a small amount (for example, an unknown volume) of blood (for example, less than 1 drop (<1 µL)) from the finger to the CROF Attach directly to the card, close the card, insert the card into the optical adapter, and finally use the smartphone to take a picture of the card. From the pictures taken, the software analyzes and gives blood counts and other parameters. The total time from attaching blood to the CROF card to displaying the blood cell count is 12 to 19 seconds on the smartphone. It is 1 to 2 seconds for attaching blood to the CROF card, and for closing the card. 3-5 seconds, 2-4 seconds to insert the card into the adapter, 3-5 seconds to take an image, and 3 seconds to finish the analysis and display blood count results. is there.

One important innovation of BCI is the CROF card technology [Ref] developed by the present inventors. The CROF card contains two thin plastics, each about 1 inch by 1 inch in area, one sheet thick and hinged to another plastic at one edge (Figure 30). (Note that a hinged connection is not necessary, but it is convenient). The CROF card is (i) an important area (~500 mm ² ) confined by the two plates of the CROF during processing of the blood sample, from the attached shape (eg pool of 2 mm diameter, 0.4 mm height). The ability to quickly (eg, 1 second) spread a blood sample onto a uniform film with a thickness of 2 μm (about 1/200 of the original thickness), and (ii) reach a thickness of 2 μm The ability to stop further reduction and (iii) the ability to hold a uniform 2 um thickness when the hand is released from the press (ie self-holding due to capillary forces between blood and plate). , (Iv) with such a thin thickness, preventing evaporation of the sample (ie by being confined by two plates evaporation occurs only at the edges of the blood membrane and for a long time in the examination area of the sample). It has an important function of zero evaporation). Empirically, using optical interference (ie, the Fabry-Perot effect from the two inner surfaces of the CROF card), we found that a CROF card from the Essenlix company has an area of at least 20 mm×20 mm, It has been discovered that with a% uniformity (ie, 100 nm), a uniform thickness of 2 μm can be retained.

  Compared to conventional methods, the CROF card offers some important and unprecedented advantages for blood cell counting. The most striking advantage is, as we have observed, when the blood drop reforms to a thickness of only one red blood cell (about 2 um) and is confined to a uniform blood layer between the two plates. , (I) blood cells in fresh undiluted whole blood without the addition of anticoagulant are well separated from each other, do not clot, have much less movement of blood cells and are easily recognizable by imaging, (Ii) The blood sample includes substantially zero evaporation in the test area, thereby maintaining a constant blood cell concentration for an extended period of time.

  The second important advantage of the CROF card is the "automatic" measurement of the volume of the blood sample (since the sample thickness is determined). A third important advantage is the use of a minimum amount of blood sample (because there is no fluid inlet or outlet, or any sample transfer channel and/or device). Other advantages are (i) being able to mix the sample with the dry reagent on the surface of the CROF card in a few seconds, (ii) simple and fast, being manually operated, and (iii) convenient and costly. Is low.

  The method of blood cell counting by imaging a blood sample trapped between two plates has a history of over 150 years and is the basis of commercially available manual hemocytometers, but to our knowledge, Has never performed a hemocytometer using a blood layer confined to a plate of uniform thickness, such as one red blood cell thickness, and no one is at or near one red blood cell thickness We have not tested blood cell behavior on homogeneous confined blood samples. In conventional imaging-based methods, the blood sample confinement interval is greater than the red blood cell thickness, so the blood sample is diluted (always use anticoagulant) to avoid red blood cell overlap (and thus miscounting). There is a need. In our study, the interest of blood cells in whole blood samples confined between two plates and having a uniform sample thickness of one red blood cell thickness or slightly greater or less than that Observe the behavior. Depending on the confinement gap (ie sample thickness) of the CROF card, the behavior of blood cells will differ significantly.

  We first observe fresh undiluted whole blood without anticoagulant added from the pricked finger to the CROF card (FIG. 31.a). For a 2 um confinement gap, light microscopy images show that all blood cells (RBC, WBC, PLT) are separated (ie, non-overlapping) from each other in the plane of the sample, and each RBC is shaded. It has a well-defined boundary surrounding each cell with a center, and each boundary does not intersect the boundaries of other RBCs. Furthermore, during the imaging period, almost no cell movement is observed. One explanation for this behavior is that the 2 um confinement gap is slightly smaller than the average thickness of red blood cells, and each RBC is slightly pinched by the confinement plates, leaving no room for other cells to move and therefore migrate. Is something that cannot be done. Clearly, the behavior of cells in the 2 um gap provides optimal conditions for cell counting by imaging.

  However, at the 2.2 um gap, some RBCs begin to overlap with other RBCs, but do not observe platelet overlap. A possible reason is that there is not enough space for platelets to overlap PLT. At the 2.6 um and 3 um gaps, more RBCs overlap, the overlap of three RBCs becomes visible, and platelets overlap with RBCs. These overlaps increase with the gap. Blood cell counts from these imagings are possible with 2.2 um, 2.6 um, and 3 um gaps, but accuracy decreases with increasing gap. At the 5um and 10um gaps, large numbers of cells overlap (eg, coagulate), RBC overlap is visible, and many RBCs have a narrow elliptical shape due to rotation of the RBC relative to the imaging plane (large gap allows rotation). Have. Clearly, accurate counting of blood cells in these gaps is very difficult, if not impossible.

  When we observe here the undiluted whole blood containing the stored anticoagulant (subjects collected by a commercial service (Bioreclamation company)), our study (Fig. 31.b) According to it, it has a different response to the confinement gap of the CROF card compared to fresh undiluted blood without anticoagulant added. In the case of a 2 um gap, the blood cell behavior in the stored blood is similar to that of fresh blood without anticoagulant added. However, for larger gaps, the stored anticoagulant-containing blood has a different 2D imaging behavior than fresh blood without anticoagulant added. In the case of a 2 um confinement gap containing anticoagulant, the RBCs do not coagulate but (a) can overlap each other on top of each other and (b) rotate in a narrow ellipse in a 2D plane image, both of which are The cell counting accuracy can be significantly reduced.

  In the blood cell counting by the smartphone BCI shown here, the confinement gap of the CROF card (thus the sample thickness) is preset to 2 um with an accuracy higher than 5%. The sample volume is determined by the sample thickness preset by the CROF card and the image of the relevant area taken by the smartphone. Blood concentration (RBC, WBC, PLT) is determined by counting cells in the relevant area from the image taken with the smartphone and dividing by the relevant volume. The RBC mean red blood cell volume (MCV) is determined by measuring the area of each RBC of the 2D planar image and the average total volume associated with each RBC using a preset sample thickness of 2 μm. The hematocrit value is determined from the product of MCV and RBC concentration.

  To count the three WBC differences (granulocytes, lymphocytes, monocytes), we placed a layer of dried acridine orange (AO) dye on the surface of one of the CROF cards to Stain the sample. Since AO stains nucleic acid and stains DNA and RNA differently, it stains only WBC and PLT and stains differently according to the number and ratio of DNA and RNA in each cell, and does not stain RBC. .. The difference in staining yielded different fluorescence wavelengths (eg, green emission at 525 nm for stained DNA and red emission at 685 nm for stained RNA) and intensities, resulting in three WBC differences. It is possible to recognize each of them and PLT. We have found that when using a CROF card, WBCs are stained by a pre-applied AO dye layer in less than 5 seconds due to the small sample thickness and thus the short dye diffusion time. In addition to brightfield microscopy for dye staining of WBCs and their fluorescence donation, another method of measuring WBCs is used in the following validations.

  The optical adapter allows an effective field of view of a circle of 0.84 mm x 0.63 mm for RBC, 2.8 mm x 2.1 mm for WBC and a radius of 0.2 mm for PLT. Currently, the optical adapter needs to move within the slider to separate the RBC and WBC, adding about 5 seconds of operating time. In the next generation, compound optical adapters that do not require sliders will be developed. All software for image analysis, user interfaces and iPhone controls is custom coded and built using specific open source code. Currently, all blood cell analyzes presented herein are completed by our software in less than 2 seconds to count blood cells from images, except for PLT analysis which will be less than 5 seconds in the next generation.

  To verify the smartphone BCI, we compare it with the following four different reference methods (RM). The RM-1 uses a high resolution microscope (Nikon Dia Photo Inversion Microscope), a non-iphone DSLR camera, and an optical adapter to read the CROF card in the same reading area as the current optical adapter iphone BCI. The RM-2 is different except that the reading area of the CROF card extends to a 3×3 array with a period of 8 mm (9 reading areas in total) and is evenly distributed in the area of the 16 mm×16 mm CROF card. Same as RM-1. RM-3 is imaged using a commercially available manual hemacytometer (purchased from Sigma-Aldrich, Z359629) and the same microscope and camera as RM1 and 2, but the imaging area is 3 mm×3 mm. The manual hemacytometer has two specific chambers, each measurement area is 3 mm×3 mm and the gap is 100 μm. In order to measure PLT, 100-fold blood dilution and RBC lysis are required. The RM-4 uses a commercial PoC hemocytometer (manufactured by one of the largest blood test equipment companies), which uses flow cytometry, and is approximately 1 cubic foot in size and weighs approximately 20 pounds. , The cost is about $20,000. The PoC device requires at least 10 μL of blood (10 drops or more), blood diluent, 3 liquid reagents (lysis, dilution and wash), 5 minutes operating time and 30 minutes daily calibration. The comparison makes it possible to examine the individual functions and the combined functions of the CROF card, the optical adapter and the imaging of the smartphone and the imaging of the microscope in relation to their performance in blood cell counting.

  For validation, (i) pooled blood purchased from a commercial vendor (Boreclamation company) and mixed with anticoagulant (EDTA), and (ii) fresh blood taken from the fingers of two volunteers ( In each test, fresh blood drawn from the finger is immediately applied directly from the finger to (a) a CROF card for the CROF card test, and (b) EDTA-coated tubes for commercial PoC and manual hemocytometers. ) Two types of blood are used. A total of 42 samples were tested for each method over several days.

  A total of 24 samples were tested on different 4 days (3, 3, 3, and 15 samples) and blood samples were of the same lot for the first 3 days, but for the last day They are in different lots. A total of 18 samples (6, 6 and 6 samples) were tested on fresh blood samples for different 3 days.

  The test results show many important facts. (1) For a given blood sample, in a daily perspective projection CV (variation coefficient, ratio of standard deviation to average value), the daily average value of the blood cell count of a smartphone BCI (p-BCI) and all four reference methods Match each other.

  (2) Comparing with p-BCI and RM-1, the blood cell count using the iPhone and the optical adapter developed by the present inventors for a predetermined CROF card sample was measured using a high resolution microscope and a DSLR camera. It has the same precision (CV) as used (eg, both have about 12% CV for RBC) (FIG. 32).

  (3) According to the comparison of RM-1 and RM-2, the multi-field imaging of CROF card provides higher accuracy than the current single-field imaging. For RBC, CV improves from about 12% to about 6%. The multi-field display function is implemented in the next generation smartphone BCI for higher accuracy.

  (4) Comparison of RM-2 and RM-3 shows that (i) the CROF card is not only easier to use, but also has the same or better cell counting accuracy as the commercially available manual hemocytometer in blood cell counting. And in view of the facts of (ii) (i) and the comparison of RM1 and RM2, the multi-field smartphone BCI should have the same or better accuracy in blood cell counting as a commercial manual hemacytometer. There is a conclusion that there is. It is further pointed out that while the current CROF card and the manual hemacytometer are the same, the variability arises for a variety of reasons. For hemocytometers this variation results from dilution, lysis and manual counting, whereas for CROF cards the current variation (~7% for RBC) is mainly the variation in sample thickness (~5%). Resulting from, which can be further improved.

  (5) The smartphone BCI can identify each of the three WBC differences by staining and measure the intensity ratio of each WBC cell as a function of the ratio of the green fluorescence intensity to the red fluorescence intensity. The standard deviation is similar to that of other blood cell counts. It shows that each subtype of white blood cells has a specific ratio of fluorescent colors (depending on their relative amounts of RNA (red fluorescence) and DNA (green fluorescence)), and that granulocytes have large amounts of RNA and granules (hence red fluorescence). High and low green fluorescence), lymphocytes have low amounts of RNA and high amounts of DNA (hence low red fluorescence and high green fluorescence), and monocytes between granulocytes and lymphocytes. This is because it has a ratio of red fluorescence and green fluorescence.

  (6) In statistical significance, the daily variability (ie, daily) of all five methods tested was essentially the same, which was very high over the multiple day period during which the smart BCI performed the test. Indicates stable.

  Finally, (7) using current optical imaging hardware and software, imaging blood cell counts are not as accurate as commercial flow cytometry PoC devices (eg, about 7% and 1% for RBCs). However, due to its current accuracy, the p-BCI demonstrated here has significant health surveillance value and clinical value in remote and developing countries, and (ii) better accuracy. In order to obtain, it is necessary to recognize two important facts that the accuracy of p-BCI can be further improved. Clearly, BCI technology has wide and important applications in cell imaging, immunological assays, nucleic acid assays, personal health monitoring and other biochemical detections.

Particular aspects of the invention are described in the following documents, which are all incorporated by reference for various purposes:
Claiming benefit of US Provisional Application No. 61/622,226 filed April 10, 2012, is a §371 application of US 2011/037455 filed May 20, 2011, and May 21, 2010. March 15, 2013, a continuation-in-part of US patent application Ser. No. 13/699,270, filed June 13, 2013, claiming benefit of filed US provisional application No. 61/347,178. Filed US Patent Application No. 13/838,600 (NSNR-003),
June 2013, which is §371 application of International Application No. US2011/037455 filed May 20, 2011, claiming the benefit of US Provisional Application No. 61/347,178 filed May 21, 2010. US Patent Application No. 13/699,270 (NSNR-001) filed on March 13, and US Provisional Application No. 61/801,424 (NSNR-004PRV) filed March 15, 2013, March 2013. Provisional Application No. 61/801,096 (NSNR-005PRV) filed on 15th March, United States Provisional Application No. 61/800,915 (NSNR-006PRV), filed March 15, 2013, March 2013 Provisional Application No. 61/793,092 (NSNR-008PRV) filed on 15th March, Provisional Application No. 61/801,933 (NSNR-009PRV), filed March 15, 2013, March 2013 Provisional US Patent Application No. 61/794,317 (NSNR-010PRV) filed on 15th March, Provisional US Patent Application No. 61/802,020 (NSNR-011PRV) filed on March 15, 2013 and March 2013 U.S. Provisional Application No. 61/802,223 filed on the 15th (NSNR-012PRV).

  Further examples of objects of the present invention are described in the enumerated paragraphs below.

  As used herein, the terms “applied” and “configured” refer to a part, component, or other object designed and/or intended to perform a given function. Point to. Thus, the use of the terms "applied" and "configured" refers to a component, component, and/or other object, rather than a given part, component, or other object "capable of" performing a function. An object refers to being selected, created, implemented, utilized, programmed, and/or designed for the purpose of performing its function. Within the scope of the present disclosure, parts, components, and/or other objects suitable for performing a particular function are additionally or alternatively described as configured to perform that function, and vice versa. Is the same. Similarly, objects that are described as configured to perform a particular function may additionally or alternatively be described as operable to perform that function.

  As used herein, when used in reference to one or more parts, features, details, structures, embodiments, and/or methods according to the present invention, the phrase "for example", the phrase "examples" As" and/or simply the terms "examples" and "exemplary" are used to describe the components, features, details, structures, embodiments and/or methods described, and the components, features according to the invention. , Details, structures, embodiments, and/or methods are not intended to be limiting. Therefore, the described parts, features, details, structures, embodiments, and/or methods are not intended to be limiting, required, and exclusive/exhaustive, but structurally and/or functionally Other features, details, structures, embodiments, and/or methods, including similar and/or equivalent parts, features, details, structures, embodiments, and/or methods, are within the scope of the disclosure.

  As used herein, the terms "at least one" and "one or more" when referring to one or more entity lists refer to any one or more of the entity lists. Moreover, the present invention is not limited to each entity specifically described in the entity list and at least one of each entity. For example, "at least one of A and B" (or equivalently "at least one of A or B" or equivalently "at least one of A and/or B") means only A, only B, or A It may be a combination with B.

  As used herein, the term "and/or" that is placed between a first entity and a second entity refers to (1) the first entity, (2) the second entity, and (3) Refers to one of the first entity and the second entity. Multiple entities listed with "and/or" should be construed in the same fashion, ie, "one or more" of the entities f so conjoined. Other entities may optionally be present, other than those specifically identified by the phrase "and/or", whether or not associated with the identified entity. Thus, as a non-limiting example, "A and/or B" is used in combination with an open-ended language such as "includes" in some embodiments (optionally includes entities other than B). Refer only to A, in certain embodiments only to B (optionally including entities other than A), and in yet another embodiment to both A and B (optionally including other entities) You can These entities may be parts, actions, structures, steps, operations, values, etc.

  Any patents, patent applications, or other references incorporated herein by reference and (1) defining terms in a conflicting manner and/or (2) incorporating the disclosure in other manners Conflicting with any portion or other incorporated reference, the unincorporated portion of the invention controls and the term or incorporated disclosure therein is the reference and/or term in which the term is defined. Or, the embedded disclosure should be controlled only with respect to the reference for which it is inherently present.

  It is believed that the following claims particularly point out novel and non-obvious specific combinations and subcombinations directed to the disclosed invention. Inventions embodied in other combinations or subcombinations of features, functions, elements, and/or characteristics may be required by amendment of the claims of the present invention or presentation of new claims in the present application or related applications. .. Such amendments or new claims may be different, wider, narrower, or different from the original invention, whether they are directed to different inventions or the same invention. It is also considered to be included within the subject matter of the disclosed invention, whether or not it is equal to the claims.

Claims (83)

(A) step of preparing a sample containing the obtained analyte,
(B) obtaining a first plate and a second plate that are movable relative to each other in different configurations, each plate having a flat sample contact surface, one or two One plate is flexible, one or two of said plates comprising spacers fixed to their respective sample contact surfaces, said spacers having a pillar shape, a flat top surface, a predetermined uniform height, and Has a predetermined constant distance between spacers that is at least twice as large as the size of the analyte, and the product of the Young's modulus of the spacer and the filling rate of the spacer is 2 MPa or more, and the filling rate is The ratio of spacer contact area to plate area, step,
(C) attaching the sample to one or two of the plates when the plates are configured in an open configuration, the open configuration wherein the two plates are partially or completely separated. And the spacing between the plates is not adjusted by the spacers,
(D) After (c), the two plates are integrated into a closed configuration, wherein at least a portion of the sample forms a layer of uniform thickness, the uniform thickness being Limited by the sample contacting surface of the plate, the uniform thickness of the layer being adjusted by the spacer and the plate, and (e) the uniform thickness while the plate is in the closed configuration. A method for analyzing a sample, comprising the step of analyzing said analyte in a layer of fish.   After step (d) and before step (e), the method further comprises removing the pressing force after the plate is in the closed configuration, after removing the pressing force, the uniform The thickness of the layer of thickness is (i) the same as the thickness of the layer of uniform thickness before removing the pressing force, and (ii) deviating less than 10% from the height of the spacer. The method of claim 1, wherein Including a first plate, a second plate, and a spacer,
i. The plates are movable relative to each other in different configurations,
ii. One or two plates are flexible,
iii. Each said plate has a sample contact area on its respective surface for contacting a sample containing an analyte,
iv. One or two of the plates includes the spacers fixed to respective sample contact areas, the spacers having a columnar shape, a flat upper surface, a predetermined uniform height, and at least the size of the analyte. Has a predetermined constant inter-spacer distance that is two times larger, at least one of the spacers is in the sample contact region, and the product of the Young's modulus of the spacer and the filling rate of the spacer is 2 MPa or more. Yes,
The fill factor is the ratio of the spacer contact area to the total plate area, and one configuration is that the two plates are separated and the spacing between the plates is not adjusted by the spacer and the sample is one of the plates. Attached to one or two, an open configuration, and another configuration is a closed configuration configured after the sample is attached in the open configuration, wherein in the closed configuration at least a portion of the sample comprises: A sample compressed by the two plates into a layer of very uniform thickness, the uniform thickness of the layers being limited by the sample contact surface of the plate and adjusted by the spacer and the plate. Device for analyzing.   The method of claim 1, wherein the distance between the spacers is in the range of 7 μm to 200 μm. The device according to claim 3 , wherein the distance between the spacers is in the range of 7 μm to 200 μm. The method according to claim 1 or 4 , wherein the flexible plate has a product of the thickness of the flexible plate and the Young's modulus of the flexible plate in the range of 60 GPa-μm to 750 GPa-μm.   The device according to claim 3 or 5, wherein the flexible plate has a product of the flexible plate thickness and the Young's modulus of the flexible plate in the range of 60 GPa-μm to 750 GPa-μm. The method of claim 1 , wherein the distance between the spacers is in the range of 7 μm to 120 μm . The device according to claim 3 , wherein the distance between the spacers is in the range of 7 μm to 120 μm. A value (ISD ⁴ /(hE)) obtained by dividing the fourth power of the distance between spacers (ISD) by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate is 1 × 10 ⁶ μm. The method according to claim 1 , 4, 6, or 8, which is ³ /GPa or less. A value (ISD ⁴ /(hE)) obtained by dividing the fourth power of the distance between spacers (ISD) by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate is 1 × 10 ⁶ μm. The device according to claim 3, 5, 7, or 9 , which is ³ /GPa or less. The method of claim 1 , 4, 6, 8, or 10 , wherein the spacer has a ratio of the lateral dimension of the spacer to the height of the spacer that is at least 1. 12. The apparatus of claim 3, 5, 7, 9, or 11 , wherein the spacer has a ratio of the lateral dimension of the spacer to the height of the spacer that is at least 1. The product of the Young's modulus of the spacer and the filling rate of the spacer is 20 MPa or more, and the filling rate is the ratio of the spacer contact area to the total plate area . Or the method described in 12 above. The product of the Young's modulus of the spacer and the filling rate of the spacer is 20 MPa or more, and the filling rate is the ratio of the spacer contact area to the total plate area . Or the apparatus according to 13 . 15. The method of claim 1 , 4 , 6, 8, 10, 12, or 14 , wherein the spacer has a fill factor of 1% or greater, the fill factor being the ratio of spacer contact area to total plate area. .. 16. The apparatus of claim 3, 5, 7, 9, 11, 13, or 15 , wherein the spacers have a fill factor of 1% or greater, and the fill factor is the ratio of spacer contact area to total plate area. .. 17. The method according to claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the spacers are arranged in a periodic array form. 18. A device according to claim 3, 5, 7, 9, 11, 13 , 15, or 17 , wherein the spacers are arranged in a periodic array configuration. The value (ISD ⁴ /(hE)) obtained by dividing the fourth power of the distance between spacers (ISD) by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate is 5×10 ⁵ μm. ³ /GPa or less, the spacer has a ratio of the lateral dimension of the spacer to the height of the spacer of at least 1, and the product of the Young's modulus of the spacer and the filling rate of the spacer is 20 MPa or more. , The spacer has a filling factor of 1% or more, and the flexible plate has a product of the thickness of the flexible plate and the Young's modulus of the flexible plate within the range of 60 GPa-μm to 750 GPa-μm. The method of claim 1 , 4 or 6 , wherein the spacers are arranged in a periodic array. The value (ISD ⁴ /(hE)) obtained by dividing the fourth power of the distance between spacers (ISD) by the thickness (h) of the flexible plate and the Young's modulus (E) of the flexible plate is 5×10 ⁵ μm. ³ /GPa or less, the spacer has a ratio of the lateral dimension of the spacer to the height of the spacer of at least 1, and the product of the Young's modulus of the spacer and the filling rate of the spacer is 20 MPa or more. , The spacer has a filling factor of 1% or more, and the flexible plate has a product of the thickness of the flexible plate and the Young's modulus of the flexible plate within the range of 60 GPa-μm to 750 GPa-μm. The device of claim 3, 5 or 7 , wherein the spacers are arranged in a periodic array. Said analyte, molecule cells, selected tissue, virus, and the nanoparticles of different shapes, the molecules Ru Oh proteins, peptides, DNA, RNA, a nucleic acid or other molecule of claim 1, 4, 6, The method according to 8, 10, 12, 14, 16, or 20 . It said analyte, molecule, cell, tissue, selected viruses, and different nanoparticles shape, the molecule Ru Oh proteins, peptides, DNA, RNA, a nucleic acid or other molecule of claim 3, 5, 7 , 9, 11, 13, 15, 17, 19, or 21 . The sample is amniotic fluid, aqueous humor, vitreous humor, whole blood, fractionated blood, plasma, or blood that is serum, breast milk, cerebrospinal fluid (CSF), earwax, ear chyme, chyme, endolymph fluid, Perilymph, feces, exhaled breath, gastric acid, gastric juice, lymph, mucus including peritoneal fluid, pleural fluid, pus, mucous secretion, saliva, exhaled breath condensate, sebum, semen, sputum, sweat The method according to claim 1 , which is a biological sample selected from synovial fluid, tear fluid, vomit, and urine. The sample is amniotic fluid, aqueous humor, vitreous humor, whole blood, fractionated blood, plasma, or blood that is serum, breast milk, cerebrospinal fluid (CSF), earwax, ear chyme, chyme, endolymph fluid, Perilymph, feces, exhaled breath, gastric acid, gastric juice, lymph, mucus including peritoneal fluid, pleural fluid, pus, mucous secretion, saliva, exhaled breath condensate, sebum, semen, sputum, sweat The device according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , which is a biological sample selected from synovial fluid, tear fluid, vomit, and urine. The sample is blood, serum, plasma, nasal swab, nasopharyngeal wash, saliva, urine, gastric juice, spinal fluid, tears, stool, mucus, sweat, ear wax, oil, glandular secretion, cerebrospinal fluid, tissue, semen. , Vaginal fluid, interstitial fluid derived from tumor tissue, ocular fluid, spinal fluid, pharyngeal swab, exhalation, hair, fingernail, skin, biopsy, placental fluid, amniotic fluid, cord blood, lymph fluid, cavity fluid, sputum, pus , Microbiota, meconium, breast milk, exhaled breath condensate, nasopharyngeal wash, nasal swab, pharyngeal swab, stool sample, hair, fingernails, earwax, tightening tissue, muscle tissue, nerve tissue, epithelial tissue, cartilage, cancerous The method according to claim 1 , 4 , 6, 8, 10, 12, 14, or 16, which is a biological sample selected from a sample and bone. The sample is blood, serum, plasma, nasal swab, nasopharyngeal wash, saliva, urine, gastric juice, spinal fluid, tears, stool, mucus, sweat, ear wax, oil, glandular secretion, cerebrospinal fluid, tissue, semen. , Vaginal fluid, interstitial fluid derived from tumor tissue, ocular fluid, spinal fluid, pharyngeal swab, exhalation, hair, fingernail, skin, biopsy, placental fluid, amniotic fluid, cord blood, lymph fluid, cavity fluid, sputum, pus , Microbiota, meconium, breast milk, exhaled breath condensate, nasopharyngeal wash, nasal swab, pharyngeal swab, stool sample, hair, fingernails, earwax, tightening tissue, muscle tissue, nerve tissue, epithelial tissue, cartilage, cancerous The device according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , which is a biological sample selected from a sample and a bone. The analyzing step (e) includes
i. Luminescence selected from photoluminescence, electroluminescence, and electrochemiluminescence,
ii. Surface Raman scattering,
iii. Electrical impedance selected from resistance, capacitance, and inductance, or iv. i. ~ Iii. 17. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16, comprising measuring any combination of. Further comprising an analyzer for analyzing the sample, the analyzer comprising:
i . Luminescence selected from photoluminescence, electroluminescence, and electrochemiluminescence,
ii. Surface Raman scattering,
iii. Electrical impedance selected from resistance, capacitance, and inductance, or iv. i. ~ Iii. It includes measuring any combination of apparatus of claim 3,5,7,9,11,13,15,17,19 or 21. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein said analyzing step (e) comprises imaging , image analysis, or counting of said analytes, or a combination thereof. .. Imaging before Symbol analyte, image analysis, or counting, or is intended for the analysis of samples containing combinations thereof, according to claim 3,5,7,9,11,13,15,17,19 or 21. The device according to 21 . 17. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 wherein the analyte is a labeled analyte. 22. The device of claim 3, 5, 7, 9, 11, 13 , 13 , 15 , 17 , 19 or 21 , wherein the analyte is a labeled analyte. The sample comprises one or more analytes and the one or two plate sample contacting surfaces comprises one or more binding sites, each binding site binding to and immobilizing a respective analyte. The method according to claim 1, 4 , 6, 8, 10, 12, 14, or 16 . The sample comprises one or more analytes and the one or two plate sample contacting surfaces comprises one or more binding sites, each binding site binding to and immobilizing a respective analyte. An apparatus according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 . The one or two plate sample contact surfaces each include one or more storage sites for storing one or more reagents, said reagents being dissolved or diffused into said sample during or after step (c). The method according to claim 1, 4 , 6, 8, 10, 12, 14, or 16 . One or two plate sample contacting surfaces each include one or more storage sites for storing one or more reagents, the reagents being in an open configuration in which the sample adheres to one or two of the plates. 22. The device of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 that dissolves or diffuses in or between the samples. The one or two plate sample contacting surfaces include one or more amplification sites, each of said one or more amplification sites provided that said analyte or said analyte label is within 500 nm of one amplification site. 17. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 wherein the signal from the analyte or the label can be amplified. The one or two plate sample contacting surfaces include one or more amplification sites, each of said one or more amplification sites provided that said analyte or said analyte label is within 500 nm of one amplification site. 22. An apparatus according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 capable of amplifying the signal from the analyte or the label. 17. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the sample is associated with viruses, fungi and bacteria from environmental samples such as water, soil, or biological samples. 22. The sample of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 wherein the sample is associated with viruses, fungi and bacteria from environmental samples such as water, soil, or biological samples. Equipment. The method according to claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the sample is a sample for detecting proteins, peptides, nucleic acids, synthetic compounds, and inorganic compounds. The device according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , wherein the sample is a sample for detection of proteins, peptides, nucleic acids, synthetic compounds, and inorganic compounds. 7. The sample is associated with infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, mental disorders, neuropsychiatric disorders, lung diseases, renal diseases, and other organic diseases . The method according to 8, 10, 12, 14, or 16 . 8. The sample is associated with infectious and parasitic diseases, injuries, cardiovascular diseases, cancer, psychiatric disorders, neuropsychiatric disorders, lung diseases, renal diseases, and other organic diseases . The device according to 9, 11, 13, 15, 17, 19, or 21 . The method according to claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the sample is a sample for detecting proteins, peptides, nucleic acids, synthetic compounds, and inorganic compounds. The device according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , wherein the sample is a sample for detection of proteins, peptides, nucleic acids, synthetic compounds, and inorganic compounds. The method according to claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the sample is a sample in the areas of human, veterinary medicine, agriculture, food, environment, and drug testing. 22. The sample according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , wherein the sample is a sample in the areas of human, veterinary medicine, agriculture, food, environment and drug testing. apparatus. 22. The device of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 further comprising a mobile communication device that communicates with a remote location via WiFi or a cellular network. 22. The device of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , further comprising a mobile communication device that is a cell phone. 22. The device of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , further comprising a mobile communication device that receives prescriptions, diagnostics, or advice from a remote medical professional. 17. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 wherein the analyte comprises stained cells. 22. The device of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , wherein the analyte comprises stained cells. Further including a plurality of scale marks, an imager, and a processing device,
(I) At least a part of the plurality of scale marks includes the spacers arranged periodically,
(Ii) the imager images the scale mark and the device,
(Iii) the processing device processes one or more images imaged by the imager,
Device according to claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19 or 21 . i. An imager for imaging the device; and
ii. Further comprising a processing device for processing one or more images from the imager,
The processing device processes the one or more images using an image processing algorithm, and the image processing algorithm is a particle number algorithm, a LUT (lookup table) filter, a particle filter, a pattern recognition algorithm, a form. 6. One or more selected from the group consisting of a geometric determination algorithm, a histogram, a line profile, a terrain representation, a binary conversion, and a color matching profile . The device according to 17, 19, or 21 . iii. An imager for imaging the device; and
iv. Further comprising a scanner configured to image different areas of the device,
22. The device of claim 3, 5, 7, 9, 11, 13 , 15 , 17 , 19, or 21 , wherein the spacer comprises a periodic array of spacers. The device further comprises a plurality of scale marks, an imager, and a processing device,
(I) At least a part of the plurality of scale marks includes the spacers arranged periodically,
(Ii) the imager images the scale mark and the device,
(Iii) The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the processing device processes one or more images imaged by the imager. The device is
i. An imager for imaging the device; and
ii. Further comprising a processing device for processing one or more images from the imager,
The processing device processes the one or more images using an image processing algorithm, and the image processing algorithm is a particle number algorithm, a LUT (lookup table) filter, a particle filter, a pattern recognition algorithm, a form. 15. A geometric determination algorithm, a histogram, a line profile, a terrain representation, a binary conversion, and one or more selected from the group consisting of color matching profiles . Or the method according to 16 . The device is
i. An imager for imaging the device; and
ii. Further comprising a scanner configured to image different areas of the device,
17. The method of claim 1 , 4 , 6, 8, 10, 12, 14, or 16 , wherein the spacer comprises a periodic array of spacers.   22. The device of claim 3, 5, 7, 9, 11, 13, 15, 17, 19, or 21, wherein the analytes include white blood cells, red blood cells, and platelets.   The spacers are arranged periodically and have a center-to-center distance between two adjacent spacers in the x direction, which is different from the case in the y direction, and the x direction is orthogonal to the y direction. , 9, 11, 13, 15, 17, 19, or 21.   22. The device of claim 3, 5, 7, 7, 9, 11, 13, 15, 17, 19, or 21 wherein the plate further comprises wells in the sample contact area.   14. The plate has different plate thicknesses at different positions of the plate, the different plate thickness controlling the sample thickness at the different positions. , 15, 17, 19, or 21.   17. The method of claim 1, 4, 6, 8, 10, 12, 14, or 16 comprising imaging cells in blood and counting the number of cells.   9. The sample thickness is reduced to reduce reagent diffusion time, and in a closed configuration, the device is configured to analyze the sample in 60 seconds or less. 17. The method according to 10, 12, 14 or 16.   The device further comprises a dry reagent applied to one or both plates, the reagent being a capture agent, a detection agent, a chemical compound, an optical label, a radioactive label, an enzyme, an antibody, a protein, a nucleic acid, a DNA, an RNA. 17. A method according to any of claims 1, 4, 6, 8, 10, 12, 14, or 16, comprising a lipid, a carbohydrate, a salt, a metal, a surfactant, a solvent, or any combination thereof.   9. The plate has multiple reagent sites and the measurement time is longer than the saturation incubation time of the assay but shorter than the time of significant interdiffusion between two adjacent sites. 17. The method according to 10, 12, 14 or 16.   17. The method of claim 1, 4, 6, 8, 10, 12, 14, or 16 wherein the analyzing step is performed without washing.   17. The method of claim 1, 4, 6, 8, 10, 12, 14, or 16, wherein the sample is not flowable.   The analyzing step uses a measuring device to detect and/or quantify the analyte by measuring a signal associated with the analyte, the signal being an optical signal, an electrical signal, a mechanical signal, a chemical signal. A method according to claim 1, 4, 6, 8, 10, 12, 14 or 16, which is a physical signal, or any combination thereof.   The analyzing step includes imaging the sample and communicating the results using a mobile communication device, wherein the mobile communication device detects (i) the sample. One or more cameras for: and (ii) a signal processor, hardware and software for receiving and/or processing detected signals and/or images of the sample and for remote communication. 17. The method according to 1, 4, 6, 8, 10, 12, 14, or 16.   The analyzing step comprises (a) imaging the sample to generate data using an imager, and (b) processing the data from the imager using a processing device. Wherein the processing comprises comparing the processed data with a database stored on the device to retrieve instructions for a course of action to be performed by the subject. The method according to 6, 8, 10, 12, 14, or 16.   The analyzing step comprises (a) using the imager to image the sample to generate data, and (b) using a processing device to process the data from the imager. 17. The method of claim 1, comprising, and said processing comprises determining the accuracy of the test.   The analyzing step includes imaging with an imager, imaging the sample includes imaging spacers with the sample, the spacers being in a periodic array, the spacers being used as scale markers. 17. The method of claim 1, 4, 6, 8, 10, 12, 14, or 16 which is performed.   4. The analyzing step comprises imaging one or more images, including imaging the spacer using an imager, the spacer being in a periodic array, the spacer being used as a scale marker. The method of claim 4, 6, 8, 10, 12, 14, or 16.   The analyzing step comprises (a) using an imager to obtain one or more images of the sample between the plates, and (b) using a scanner to scan different regions of the sample. 17. The method of claim 1, 4, 6, 8, 10, 12, 14, or 16 comprising:   17. The sample of claim 1, wherein the sample is blood and the analyte comprises one or more selected from the group consisting of white blood cells, red blood cells, and platelets. The method described.   8. The spacers are arranged in a rectangular grid array and have a height of 30 μm and a center-to-center distance between two adjacent spacers of 110 μm in the x direction and 120 μm in the y direction. , 9, 11, 13, 15, 17, 19, or 21.   The method according to any one of claims 1, 4, 6, 8, 10, 12, 14, and 16, wherein the spacer has a height of 50 μm or less.   17. The method of any one of claims 1, 4, 6, 8, 10, 12, 14, and 16, wherein the spacer has a height of 5 [mu]m or less.   The method according to any one of claims 1, 4, 6, 8, 10, 12, 14, and 16, wherein the spacers have a uniform height between 20 μm and 30 μm.   22. The device of claim 3, 5, 7, 7, 9, 11, 13, 15, 17, 19, or 21, wherein the spacers are divided into different regions, each region having a respective spacer height.

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